{"paper_id":"36cea40b-6e26-45ac-97f8-8d1798a0a3ba","body_text":"Development, characterization, and cross-amplification of polymorphic microsatellite markers for North American Trachymyrmex and Mycetomoellerius ants | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research note Development, characterization, and cross-amplification of polymorphic microsatellite markers for North American Trachymyrmex and Mycetomoellerius ants Alix E. Matthews, Chase Rowan, Colby Stone, Katrin Kellner, Jon N. Seal This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.2.21905/v2 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 24 Mar, 2020 Read the published version in BMC Research Notes → Version 2 posted 4 You are reading this latest preprint version Show more versions Abstract Objective The objective of this study is to develop and identify polymorphic microsatellite markers for fungus-gardening (attine) ants in the genus Trachymyrmex sensu lato . These ants are important ecosystem engineers and have been a model group for understanding complex symbiotic systems, but very little is understood about the intraspecific genetic patterns across most North American attine species. These microsatellite markers will help to better study intraspecific population genetic structure, gene flow, mating habits, and phylogeographic patterns in these species and potentially other congeners. Results Using next-generation sequencing techniques, we identified 17 and 12 polymorphic microsatellite markers from T. septentrionalis and Mycetomoellerius (formerly Trachymyrmex ) turrifex , respectively, and assessed the genetic diversity of each marker. We also analyzed the cross-amplification success of the T. septentrionalis markers in two other closely related Trachymyrmex species, and identified 10 and 12 polymorphic markers for T. arizonensis and T. pomonae , respectively. Population Genetics Attini fungus-gardening ant intraspecific genetic diversity microsatellites population genetics Introduction Fungus-gardening ants (tribe Attini) have been subject to decades of research elucidating their major evolutionary transitions and radiations [ 1-5 ]. Despite these revolutionary advances, most attine research has occurred at higher taxonomic levels (species or higher). Consequently, we know very little about the microevolutionary processes that may underlie macroevolutionary (phylogenetic) patterns. This lack of understanding is partially technical since most genetic markers used to construct phylogenies typically exhibit little intraspecific variation, for both the ants and fungi [ 2 , 6-8 ]. Mitochondrial DNA (mtDNA) markers, while useful for large scale geographic studies [ 9-11 ], may be generally problematic because some attines may have nuclear-incorporated mtDNA-like sequences or whose genomes contain pseudogenes [ 7 , 12 ]. Moreover, mtDNA is maternally inherited, which may or may not be indicative of intraspecific patterns of gene flow since male and female ants are known to exhibit different patterns of gene flow [ 13-15 ]. Finer-level intraspecific examinations have typically required specific diploid markers such as microsatellites or single-nucleotide polymorphisms (SNPs) [ 16-19 ]. Both approaches represent co-dominant expression and genome-wide distributions, but microsatellites are short, non-coding, highly variable repetitive motifs [ 20 ], whereas SNPs are shorter and found throughout coding and non-coding genomic regions [ 21 ]. SNPs are popular in the genomics age because of the large quantities of potentially informative sites quickly generated in a single sequencing run, but they require considerable time and effort to identify and analyze, and are typically restricted to single studies. Conversely, microsatellite markers, once developed, can be used repeatedly in multiple studies, which may reduce their overall expense on a per-project basis [ 22 ]. Moreover, recent comparative studies in insects, fungi, and birds reported no major population genetic structural differences between these two approaches [ 23-26 ], or that more SNPs were required to produce the same information as a single microsatellite marker [ 27 ]. Traditional microsatellite development techniques (i.e., constructing libraries, cloning) have yielded polymorphic microsatellite markers for both higher and lower attines [ 28-32 ]. However, because these markers were developed for tropical attines, they may or may not be useful for North American species (J.N. Seal, unpublished observations), which tend to belong to phylogenetically distinct lineages [ 3 , 33 ]. Next-generation sequencing (NGS) techniques have revolutionized microsatellite marker development in terms of decreased time and cost, and increased quantity of potential markers [ 34-36 ], although only a few studies have utilized these techniques for attines. Extensive marker sets exist for Acromyrmex lundii [ 37 ], Atta laevigata [ 38 ], and two species in the lower attine genus Mycocepurus [ 18 , 39 ]. Here, our aim was to use NGS techniques to develop microsatellite markers for two additional attine species, Trachymyrmex septentrionalis and Mycetomoellerius turrifex . Both are common and ecologically important fungus-gardening ants throughout southeastern North America [ 10 , 40 , 41 ]. We also explored the potential for cross-amplification of the T. septentrionalis markers in two closely related species: T. arizonensis and T. pomonae . These three species belong to Trachymyrmex sensu stricto , unlike M. turrifex which belongs to a new genus, Mycetomoellerius [ 3 , 33 , 42 ]. These markers will provide a new tool to study patterns of attine intraspecific genetic variation and phylogeographic structure. Methods Primer sets were developed from a single T. septentrionalis forager collected in north Florida ([ 10 ] Site C; 30.2796, -84.9004) and a single M. turrifex forager collected in central Texas (University of Texas at Austin; 30.2861, -97.7297). Illumina sequencing and primer design were conducted at the Savannah River Ecology Laboratory (University of Georgia, USA) in the Molecular Ecology Laboratory. A Covaris S220 Focused-ultrasonicator sheared 1 μg of genomic DNA to prepare an Illumina paired-end shotgun library following standard Illumina TruSeq DNA Library Kit protocols and using a multiplex identifier adaptor index. Sequencing was conducted on an Illumina HiSeq 3000 platform (San Diego, CA, USA) producing 100-bp paired-end reads. PAL_FINDER v.0.02.03 [ 43 ] was used to analyze five million resulting reads, and those with di-, tri-, tetra-, penta-, and hexanucleotide microsatellites were extracted. Positive reads were batched to a local installation of Primer3 v.2.0.0 [ 44 , 45 ] for primer design. Markers for which the primer sequence only occurred three or four times in the five million reads were selected to avoid duplication. From this selection of 9,293 ( T. septentrionalis ) and 6,943 ( M. turrifex ) markers, we chose a subset to test (46 for T. septentrionalis ; 20 for M. turrifex ) following an M13-tail PCR protocol [ 46 ]. We extracted genomic DNA from individual worker ants (from restricted portions of their respective ranges; Additional file 1) preserved in 95% ethanol at -20°C using a QIAamp DNA Micro Kit (QIAGEN). In particular, ants used in the development of T. septentrionalis markers were all collected in Florida (i.e., not Texas) (Table 1). For all species, the 10 μL PCR mix contained 1 μL of 10X PCR buffer (1.0X; Applied Biosystems), 1 μL Bioline® dNTP mix (1 mM each; 0.1 mM proportional to total), 1.5 μL of 25 mM MgCl 2 (3.75 mM; Applied Biosystems), 0.5 μL of 20 μM BSA (1 μM; New England Biolabs), 0.3 μL of 2 μM tag-labeled primer (0.06 μM; forward primer with M13-tail), 0.6 μL of a 2 μM universal dye-labeled primer (0.12 μM; FAM label with M13-tail), 1 μL of 2 μM unlabeled primer (0.2 μM; reverse primer), 0.1 μL of Taq polymerase (0.5 U; Applied Biosystems), and 1 μL of DNA template. PCR cycling conditions consisted of an initial denaturation of 4 min at 95°C, 25 (or 30) cycles of 30 s at 95°C, 45 s at the primer-specific annealing temperature identified by a temperature gradient program, 45 s at 72°C, then 8 cycles of 30 s at 95°C, 45s at 53°C, and 45 s at 72°C, and a final extension of 5 min at 72°C. PCR products were run on an Applied Biosystems 3730 Genetic Analyzer and fragments were sized with LIZ600 size standard at the University of Texas at Austin DNA Sequencing Facility. We scored alleles in Geneious v.10.2.3 [ 47 ]. For markers that resulted in high quality PCR product and exhibited polymorphism, we measured genetic variability for each species. We estimated the number of alleles per locus ( K ), observed and expected heterozygosity (H o and H e ), and the probability of identity (PI; the probability of two independent samples having the same genotype) using GenAlEx v.6.5 [ 48 , 49 ]. Sample sizes (numbers of individuals tested) for each marker are provided (Tables 1 and 2, Additional files 2 and 3). We assessed deviations from Hardy-Weinberg equilibrium (HWE) and tested for linkage disequilibrium (LD) across all pairs of markers using GENEPOP v.4.2 [ 50 ] employing Bonferroni corrections for multiple comparisons. The presence of null alleles was assessed using Microchecker [ 51 ]. Results For Trachymyrmex septentrionalis markers tested on T. septentrionalis ants, 37 of the original 46 positively amplified with 17 being reliably amplified and scored. The other 20 either showed weak amplification, weak polymorphism (2-4 alleles per marker), monomorphism, or multi-peak profiles. The number of alleles across all samples of the 17 reliable markers ranged from 9 to 33 (average = 16.2). H o varied from 0.11 to 0.89 (average = 0.62), while H e ranged from 0.65 to 0.94 (average = 0.84). After Bonferroni correction, 9 markers showed significant deviations from HWE. There were two cases of LD detected across the 136 paired markers (between Ts4 and Ts5, and Ts3 and Ts43; Table 1; Additional file 4). Most of the loci tested significantly for the presence of null alleles, which suggests that T. septentrionalis may have even higher allelic diversity than what we captured in our sampling (Table 1). Of the 20 original Mycetomoellerius turrifex markers, 17 positively amplified with 12 reliably amplified and scored. The number of alleles across all samples ranged from 4 to 12 (average = 8.9). H o varied from 0.2 to 0.71 (average = 0.49), while H e ranged from 0.58 to 0.88 (average = 0.78). After Bonferroni correction, 6 markers showed significant deviations from HWE. There were no cases of LD detected across the 66 paired markers (Table 2; Additional file 4). In contrast to T. septentrionalis , the frequency of null alleles was lower in M. turrifex (Table 2). We tested cross-species amplification of the 46 T. septentrionalis markers developed here in both T. arizonensis and T. pomonae . For T. arizonensis (n = 8), 24 markers were positively amplified with 10 reliably scored. Of those 10, all revealed at least 2 alleles, 8 revealed 3 or more alleles, and 9 exhibited some heterozygosity. There were 9 markers in HWE and one violated HWE (Ts38). There was no LD (Additional file 2; Additional file 4). For T. pomonae (n = 6), 23 markers were successfully amplified and 12 were reliably scored. Of those 12, all revealed at least 2 alleles, 8 revealed 3 or more alleles, and 10 exhibited some heterozygosity. There were 10 in HWE, one violated HWE (also Ts38), and one could not be computed (Ts7) because there was only one copy of the second allele. There was no LD (Additional file 3; Additional file 4). Only one of the loci in T. pomonae and only two in T. arizonensis appeared to have null alleles; this likely results from examining ants collected across a few hectares of pine oak forest (Southwest Research Station) (Additional Files 2 and 3). Discussion These newly developed microsatellite markers provide a promising new tool for population genetic, mating, dispersal, and phylogeographic studies of Trachymyrmex septentrionalis and Mycetomoellerius turrifex populations across their ranges. Positive cross-species amplification also indicates that some of the T. septentrionalis markers appear to be conserved in at least two other congeneric species ( T. arizonensis and T. pomonae ). This indicates that future analyses for additional individuals across a wider geographic region may be informative for understanding similar questions for T. arizonensis and T. pomonae and perhaps other closely related Trachymyrmex . It also seems likely that the markers developed here for M. turrifex should cross amplify in closely related Mycetomoellerius species, such as the highly studied M. zeteki [ 42 , 52 , 53 ], among others. While all markers across all four species showed polymorphism/allelic variation, several markers (particularly within T. septentrionalis ) were hypervariable (over 20 alleles per locus; Table 1). These hypervariable markers, collectively with other polymorphic markers, may be informative for investigating intraspecific dynamics at an especially fine-scale (e.g., mating habits such as multiple-mating or multiple-queen hypotheses by comparing relatedness of individuals within colonies). Despite the extensive polymorphism illustrated by these markers, evidence showing the presence of null alleles suggests that more alleles may remain to be discovered. The frequency of null alleles may be more pronounced in populations that already demonstrate significant genetic structure [ 54 ]. The new markers developed here may also be used in conjunction with mtDNA to better understand dispersal, signatures of population expansion or contraction, and phylogeographic history. For example, male and female ants of several species may mediate gene flow differentially through sex-specific dispersal patterns [ 13 , 14 , 55-57 ], but this has yet been tested in Trachymyrmex or Mycetomoellerius ants. By employing marker systems with different modes of inheritance, we may be able to discern how ecological dispersal patterns influence ant genetic structure across populations. In a phylogeographic context, the application of both maternally and biparentally inherited markers may also help to uncover and track the progression of previously unrecognized or poorly understood historical processes such as species origin, divergence, colonization, and genetic drift. For example, the extensive evidence for null alleles in T. septentrionalis is likely driven by phylogeographic structure in this species across the relatively restricted range examined in this study (northern Florida) [ 10 ]. We explored the 17 T. septentrionalis markers across a small subset (total n = 12) of T. septentrionalis ants from Texas as compared to a random subset of T. septentrionalis ants from Florida. The allelic diversity appears to be much higher in Florida than in Texas (see Additional file 5), which may suggest some fundamental genomic differences in populations east and west of the Mississippi River. These preliminary results align with previous mtDNA results that suggest 1) a significant divergence between populations on either side of the Mississippi River and 2) limited genetic diversity west of the Mississippi River [ 10 , 11 ]. Future analyses comparing the allelic diversity of populations east and west of the Mississippi River would be insightful for better understanding these patterns. Limitations The restricted sample size and geographic distribution for arizonensis and T. pomonae may underestimate potential polymorphic markers in these species. Markers not deviating from HWE in our initial screenings could potentially deviate in other populations, which may reduce the number of usable markers in certain genetic studies; on the other hand, markers deviating from HWE in the studied populations might not deviate in other populations, which would increase the number of markers. The presence of null alleles could be a result of potential phylogeographic structure in the populations studied (especially septentrionalis ), as a result polymorphism may be underestimated. Sample sizes per marker for the Texas septentrionalis subset were smaller compared to Florida, which may limit our interpretations; however, reduced microsatellite diversity would be consistent with lower mtDNA diversity in western populations (Texas). Abbreviations bp: base pair; H e : expected heterozygosity; H o : observed heterozygosity; HWE: Hardy-Weinberg equilibrium; K: number of alleles; LD: linkage disequilibrium; mtDNA: mitochondrial DNA; NGS: next-generation sequencing; PCR: polymerase chain reaction; PI: probability of identity; SNPs: single-nucleotide polymorphisms; T m : annealing temperature Declarations Acknowledgements Larry Gilbert, Steven Gibson and Rob Plowes facilitated collections at the University of Texas at Austin Stengl Biological Station and Brackenridge Field Laboratory. Joshua King and Andrea Lucky provided T. septentrionalis specimens from the Orlando and Gainesville, Florida areas, respectively. We thank Geoff Bender, Michele Lanan and Southwestern Research Station staff for providing logistical support and facilitating collection permits in the Coronado National Forest. This is paper No. 3 of the Ant Symbiosis Laboratory. Authors’ contributions JNS and KK conceived the study, secured funding and permits, and collected samples. AEM, CR, and CS performed DNA extractions, PCR work, and tested/optimized primer pairs on all species. AEM scored alleles and conducted data analysis. AEM and JNS led manuscript writing, and all authors read, edited, and approved the final manuscript. Availability of data and material The datasets analyzed during the current study are not presently public due to their use in ongoing publication, but the genotypic data for each sample are available upon reasonable request to the corresponding author. Competing interests The authors declare they have no competing interests. Consent for publication Not applicable. Ethics approval and consent to participate Not applicable. The current research did not involve human subjects, human material, or human data or animals. 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Population Ecology 2013, 55 (4):523-533. Sanetra M, Crozier RH: Patterns of population subdivision and gene flow in the ant Nothomyrmecia macrops reflected in microsatellite and mitochondrial DNA markers . Mol Ecol 2003, 12 (9):2281-2295. Doums C, Cabrera H, Peeters C: Population genetic structure and male-biased dispersal in the queenless ant Diacamma cyaneiventre . Molecular Ecology 2002, 11 (11):2251-2264. Tables Table 1. Trachymyrmex septentrionalis polymorphic microsatellite markers analyzed for Trachymyrmex septentrionalis ants. Marker Primer sequence 5' -> 3' Repeat motif T m ( ° C) Size (bp) N K H o H e PI Ts3 a,b,c F: GCGCGGTTGTTCTTTATGG ATTTT 60.5 264-292 25 12 0.68 0.88 0.03 R: TTGTATTCTTTGTCATACAGTACGTTGC Ts4 b,c F: CTTTGAAATCGTCATCGCGG TCGGC 57.3 242-266 25 11 0.56 0.87 0.03 R: ACGCCCACACGTATACCACC Ts5 a,c F: CCACCTTGGTAACTGTCGCC TCGGC 60.5 230-256 48 11 0.67 0.81 0.06 R: CTTTGAAATCGTCATCGCGG Ts11 b,c F: GCAGATACAAACGTCCTACGTGC TGCG 66.4 274-358 20 13 0.45 0.91 0.02 R: CGCACATTTGTGACGGACG Ts12 b,c F: ATTCCTGGCACGGATACACG ATAC 61.3 164-214 26 9 0.35 0.65 0.16 R: ACTCTATTGTTGCGCACCGC Ts21 c F: CCATCCCAACCATCCTGG AGCC 65.2 302-326 45 11 0.76 0.82 0.05 R: TTACGATCAGGAGAGCGTGC Ts25 c F: CGTAAATTAAAGTGCACAGTCCG TCTG 61.3 286-346 51 18 0.78 0.86 0.03 R: GATCGCGATTTGGTGATACG Ts32 b,c F: ATAACAAGCGGCAGCATCG TTGG 59.4 200-242 49 15 0.76 0.85 0.04 R: ATTTCGAACTCGCCGGTAGC Ts33 F: AATCAAATGCTTGCGTGTGC TGCG 60.5 248-296 51 15 0.71 0.73 0.09 R: CCGGTTAGAAGAAACAGGCG Ts34 b,c F: GTGAGGGAATGAGAGGGAGG TGCG 61.3 246-490 27 12 0.11 0.81 0.06 R: TGGTAATGATCGGTACATGATGC Ts35 b,c F: TGCTCGATTCGGACACGG ACCG 60.5 188-300 49 14 0.89 0.76 0.08 R: CTCACAGCGGAGACAAAGGC Ts36 b,c F: TGTAGGGATTTAGATGCGGC TGCG 59.4 166-210 13 9 0.15 0.85 0.04 R: TAGCCAGACCTCGTACTTCG Ts39 c F: CTAACAAGATGCGCAGCCC TGCG 61.3 234-350 50 30 0.84 0.94 0.01 R: TCGAATAATCCAGTCGTGTCG Ts41 F: TTAACGTCGGCATAATTTCGG TGCC 61.3 206-236 46 14 0.78 0.82 0.05 R: CAATTGACTACGCAGGAGCG Ts43 c F: CGTCTTTATATTGTATTTGCTTGATACGC TGCG 61.3 246-358 47 33 0.72 0.94 0.01 R: GTCCATGCACACGTCCAGC Ts45 b,c F: CGTGTCAAGTATGTTCCCGC TGCG 61.3 174-316 48 28 0.60 0.89 0.02 R: AGTTTCAGGCGCAGGTAGC Ts46 c F: GTACGCACATCGTGCTAAACG ACCG 60.5 310-348 46 20 0.78 0.92 0.01 R: AGCGGTGGTGGTTTCACG Details include: marker information, primer sequences, repeat motif, annealing temperature (T m ), size range of observed alleles given in base pairs (bp), number of individuals genotyped (N), number of alleles observed (K), observed heterozygosity (H o ), expected heterozygosity (H e ), and probability of identity (PI). Superscript a indicates that 30 cycles were used in the PCR profile, superscript b indicates deviation from Hardy-Weinberg expectations after Bonferroni corrections, and superscript c indicates the marker shows evidence of null alleles. Table 2. Mycetomoellerius turrifex polymorphic microsatellite markers analyzed for Mycetomoellerius turrifex ants. Marker Primer sequence 5' -> 3' Repeat motif T m ( ° C) Size (bp) N K H o H e PI Tt5 F: ACGGAATGTGTTAACGTGCG TGCG 60.5 254-322 14 10 0.71 0.86 0.04 R: TATGTGCTCGTCGTTCTCGC Tt10 b,c F: ACCGGAGAGCGGTAGAGACC AGGT 58.3 286-320 16 11 0.5 0.85 0.04 R: TACCCGGCCATTAGAACTCC Tt18 F: GGTGATGGTCGATAGTTTCCG AACG 60.5 196-244 16 7 0.44 0.58 0.20 R: ACGACGTATGGGTTCGTGC Tt20 F: ATGCAGAGTCAGAGGACCGC TGCG 60.5 218-236 15 7 0.67 0.78 0.08 R: GAATTGTCTCCACATTATCAAGGG Tt2 b,c F: AATGTCGGACGTTTATGGTCG TTGCG 56.2 266-314 16 7 0.44 0.77 0.09 R: GTATCATCGGCACTGCAACG Tt7 a,b,c F: GCAGTATGACTTCTGATCCTTTCG TGCG 66.2 156-176 15 4 0.20 0.58 0.26 R: CACGTTAATCCAGCACTCCG Tt9 a F: ACGCACTGTGTATGTGTGCG TGCG 66.2 188-264 10 12 0.70 0.83 0.04 R: AGCATATAAGTACGAATAACTGAGATTGG Tt16 F: TCGATTTATTAGAAAGGCTTGCG AGTG 55.1 272-288 13 5 0.54 0.74 0.11 R: TGCACGAGAGTGTTTGTAGCC Tt6 b,c F: TTATATCGATGGCTTCCCACC AGGT 57.3 318-358 15 11 0.27 0.85 0.04 R: CCCTCTCGATATCTACTCGGTACG Tt14 a F: AAGTCGCGTAATGACGATGC ATAC 57.3 256-378 15 10 0.60 0.77 0.08 R: GAGATATACCTGATTCAACGTCGC Tt15 a,b,c F: TGCCTTCATATATGTGCCTTCG TGCG 65.3 238-304 12 12 0.42 0.88 0.03 R: TGTCGTTAAGAGTTACAGAACAGGG Tt17 b,c F: TCACTCAAATCGAATATGTAGATGAGG TGCG 64.4 148-198 15 11 0.47 0.88 0.03 R: CCCACAGTAATGTCCTAGTAATGTCC Details include: marker information, primer sequences, repeat motif, annealing temperature (T m ), size range of observed alleles given in base pairs (bp), number of individuals genotyped (N), number of alleles observed (K), observed heterozygosity (H o ), expected heterozygosity (H e ), and probability of identity (PI). Superscript a indicates that 30 cycles were used in the PCR profile, superscript b indicates deviation from Hardy-Weinberg expectations after Bonferroni corrections, and superscript c indicates the marker shows evidence of null alleles. Additional File Legends Additional file 1 [AdditionalFile1.docx]. List of individual ant samples used in this study. The table includes individual sample name, host species, state of collection, general sampling location, and coordinates of collection location (unless the property was under private ownership). Additional file 2 [AdditionalFile2.docx]. Details of the 10 Trachymyrmex septentrionalis polymorphic microsatellite markers analyzed for cross-amplification in Trachymyrmex arizonensis . Additional file 3 [AdditionalFile3.docx]. Details of the 12 Trachymyrmex septentrionalis polymorphic microsatellite markers analyzed for cross-amplification in Trachymyrmex pomonae . Additional file 4 [AdditionalFile4.docx]. Pairwise comparison results of the linkage disequilibrium analyses for all four species. Additional file 5 [AdditionalFile5.docx]. Details of the 17 Trachymyrmex septentrionalis polymorphic microsatellite markers analyzed for Trachymyrmex septentrionalis ants across Texas (TX) and Florida (FL), highlighting the differences between two distant populations. Details include: marker name, size range of observed alleles given in base pairs (bp), number of individuals genotyped (N), number of alleles observed (K). Supplementary Files AdditionalFile4.docx AdditionalFile1.docx AdditionalFile5.docx AdditionalFile2.docx AdditionalFile3.docx BMCResearchNotescoverletter11012020.pdf Cite Share Download PDF Status: Published Journal Publication published 24 Mar, 2020 Read the published version in BMC Research Notes → Version 2 posted Editorial decision: Accept 10 Mar, 2020 Editor assigned by journal 05 Mar, 2020 Submission checks completed at journal 04 Mar, 2020 Editor invited by journal 04 Mar, 2020 You are reading this latest preprint version Show more versions Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {\"props\":{\"pageProps\":{\"initialData\":{\"identity\":\"rs-12487\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":false,\"archivedVersions\":[],\"articleType\":\"Research note\",\"associatedPublications\":[],\"authors\":[{\"id\":397744,\"identity\":\"20432c1c-15f1-4ef6-99fe-79c012d45061\",\"order_by\":1,\"name\":\"Alix E. 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Seal\",\"email\":\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA50lEQVRIiWNgGAWjYJACZjjrAxCzsRNQzoOshXEGSAszbtWYWph5UC3FDuylDz/8XNi2LXH7tMPHpG1+bZPnY2Zg/PAxB48tfGnG0jPbbifOuZ2WbJzbd9uwjZmBWXLmNjxaeBjMmHmBWmZI5xg+zu25zQjUwsbMi1cL+zeolvwPhy17btsToYUHbgvjY4YftxMJaznDUyzNc+628QzpNGPD3obbyW3MjM14/cLew77xM0/ZbdkZ0snPJH78uW07v7354IePeLSAASMbg2MDmNEGJhsIqAeBPwz2MMYoGAWjYBSMAgwAAMPAS2B4N9exAAAAAElFTkSuQmCC\",\"orcid\":\"https://orcid.org/0000-0001-8013-1438\",\"institution\":\"\",\"correspondingAuthor\":true,\"submittingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Jon\",\"middleName\":\"N.\",\"lastName\":\"Seal\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2020-01-23 15:27:55\",\"currentVersionCode\":2,\"declarations\":\"\",\"doi\":\"10.21203/rs.2.21905/v2\",\"doiUrl\":\"https://doi.org/10.21203/rs.2.21905/v2\",\"draftVersion\":[],\"editorialEvents\":[{\"content\":\"https://doi.org/10.1186/s13104-020-05015-3\",\"type\":\"published\",\"date\":\"2020-03-24T21:11:03+00:00\"}],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":13492736,\"identity\":\"f5acf796-0263-4575-8561-84469e5658ca\",\"added_by\":\"auto\",\"created_at\":\"2021-09-16 22:32:48\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":1590884,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-12487/v2/a0669011-796a-427e-b272-e9632142718b.pdf\"},{\"id\":616860,\"identity\":\"9e179662-3b48-43ef-a1ac-7f7f6523a0fe\",\"added_by\":\"auto\",\"created_at\":\"2020-03-09 18:56:00\",\"extension\":\"docx\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":158474,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"AdditionalFile4.docx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-12487/v2/AdditionalFile4.docx\"},{\"id\":616859,\"identity\":\"8c5cffdc-eeaa-4b38-9b16-9cc920d84317\",\"added_by\":\"auto\",\"created_at\":\"2020-03-09 18:56:00\",\"extension\":\"docx\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":149693,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"AdditionalFile1.docx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-12487/v2/AdditionalFile1.docx\"},{\"id\":616858,\"identity\":\"0cedb6b4-30c9-409e-a3ac-8d799d2ebdd8\",\"added_by\":\"auto\",\"created_at\":\"2020-03-09 18:56:00\",\"extension\":\"docx\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":73844,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"AdditionalFile5.docx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-12487/v2/AdditionalFile5.docx\"},{\"id\":616857,\"identity\":\"cb1140ee-42c8-40e9-84c9-9578229a9f00\",\"added_by\":\"auto\",\"created_at\":\"2020-03-09 18:56:00\",\"extension\":\"docx\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":102176,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"AdditionalFile2.docx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-12487/v2/AdditionalFile2.docx\"},{\"id\":616856,\"identity\":\"389f9612-a68d-4c63-bddf-80b095c7e354\",\"added_by\":\"auto\",\"created_at\":\"2020-03-09 18:56:00\",\"extension\":\"docx\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":110323,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"AdditionalFile3.docx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-12487/v2/AdditionalFile3.docx\"},{\"id\":616855,\"identity\":\"43badce0-e1c0-48f0-b336-b424f9eb5fd5\",\"added_by\":\"auto\",\"created_at\":\"2020-03-09 18:56:00\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":230217,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"BMCResearchNotescoverletter11012020.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-12487/v2/BMC_Research_Notes_coverletter_11012020.pdf\"}],\"financialInterests\":\"\",\"formattedTitle\":\"Development, characterization, and cross-amplification of polymorphic microsatellite markers for North American Trachymyrmex and Mycetomoellerius ants\",\"fulltext\":[{\"header\":\"Introduction\",\"content\":\"\\u003cp\\u003eFungus-gardening ants (tribe Attini) have been subject to decades of research elucidating their major evolutionary transitions and radiations [\\u003ca href=\\\"#_ENREF_1\\\"\\u003e1-5\\u003c/a\\u003e]. Despite these revolutionary advances, most attine research has occurred at higher taxonomic levels (species or higher). Consequently, we know very little about the microevolutionary processes that may underlie macroevolutionary (phylogenetic) patterns. This lack of understanding is partially technical since most genetic markers used to construct phylogenies typically exhibit little intraspecific variation, for both the ants and fungi [\\u003ca href=\\\"#_ENREF_2\\\"\\u003e2\\u003c/a\\u003e, \\u003ca href=\\\"#_ENREF_6\\\"\\u003e6-8\\u003c/a\\u003e]. Mitochondrial DNA (mtDNA) markers, while useful for large scale geographic studies [\\u003ca href=\\\"#_ENREF_9\\\"\\u003e9-11\\u003c/a\\u003e], may be generally problematic because some attines may have nuclear-incorporated mtDNA-like sequences or whose genomes contain pseudogenes [\\u003ca href=\\\"#_ENREF_7\\\"\\u003e7\\u003c/a\\u003e, \\u003ca href=\\\"#_ENREF_12\\\"\\u003e12\\u003c/a\\u003e]. Moreover, mtDNA is maternally inherited, which may or may not be indicative of intraspecific patterns of gene flow since male and female ants are known to exhibit different patterns of gene flow [\\u003ca href=\\\"#_ENREF_13\\\"\\u003e13-15\\u003c/a\\u003e]. Finer-level intraspecific examinations have typically required specific diploid markers such as microsatellites or single-nucleotide polymorphisms (SNPs) [\\u003ca href=\\\"#_ENREF_16\\\"\\u003e16-19\\u003c/a\\u003e]. Both approaches represent co-dominant expression and genome-wide distributions, but microsatellites are short, non-coding, highly variable repetitive motifs [\\u003ca href=\\\"#_ENREF_20\\\"\\u003e20\\u003c/a\\u003e], whereas SNPs are shorter and found throughout coding and non-coding genomic regions [\\u003ca href=\\\"#_ENREF_21\\\"\\u003e21\\u003c/a\\u003e].\\u003c/p\\u003e\\n\\u003cp\\u003eSNPs are popular in the genomics age because of the large quantities of potentially informative sites quickly generated in a single sequencing run, but they require considerable time and effort to identify and analyze, and are typically restricted to single studies. Conversely, microsatellite markers, once developed, can be used repeatedly in multiple studies, which may reduce their overall expense on a per-project basis [\\u003ca href=\\\"#_ENREF_22\\\"\\u003e22\\u003c/a\\u003e]. Moreover, recent comparative studies in insects, fungi, and birds reported no major population genetic structural differences between these two approaches [\\u003ca href=\\\"#_ENREF_23\\\"\\u003e23-26\\u003c/a\\u003e], or that more SNPs were required to produce the same information as a single microsatellite marker [\\u003ca href=\\\"#_ENREF_27\\\"\\u003e27\\u003c/a\\u003e].\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eTraditional microsatellite development techniques (i.e., constructing libraries, cloning) have yielded polymorphic microsatellite markers for both higher and lower attines [\\u003ca href=\\\"#_ENREF_28\\\"\\u003e28-32\\u003c/a\\u003e]. However, because these markers were developed for tropical attines, they may or may not be useful for North American species (J.N. Seal, unpublished observations), which tend to belong to phylogenetically distinct lineages [\\u003ca href=\\\"#_ENREF_3\\\"\\u003e3\\u003c/a\\u003e, \\u003ca href=\\\"#_ENREF_33\\\"\\u003e33\\u003c/a\\u003e]. Next-generation sequencing (NGS) techniques have revolutionized microsatellite marker development in terms of decreased time and cost, and increased quantity of potential markers [\\u003ca href=\\\"#_ENREF_34\\\"\\u003e34-36\\u003c/a\\u003e], although only a few studies have utilized these techniques for attines. Extensive marker sets exist for \\u003cem\\u003eAcromyrmex\\u003c/em\\u003e \\u003cem\\u003elundii\\u003c/em\\u003e [\\u003ca href=\\\"#_ENREF_37\\\"\\u003e37\\u003c/a\\u003e], \\u003cem\\u003eAtta laevigata\\u003c/em\\u003e [\\u003ca href=\\\"#_ENREF_38\\\"\\u003e38\\u003c/a\\u003e], and two species in the lower attine genus \\u003cem\\u003eMycocepurus\\u003c/em\\u003e [\\u003ca href=\\\"#_ENREF_18\\\"\\u003e18\\u003c/a\\u003e, \\u003ca href=\\\"#_ENREF_39\\\"\\u003e39\\u003c/a\\u003e].\\u003c/p\\u003e\\n\\u003cp\\u003eHere, our aim was to use NGS techniques to develop microsatellite markers for two additional attine species, \\u003cem\\u003eTrachymyrmex septentrionalis \\u003c/em\\u003eand \\u003cem\\u003eMycetomoellerius turrifex\\u003c/em\\u003e. Both are common and ecologically important fungus-gardening ants throughout southeastern North America [\\u003ca href=\\\"#_ENREF_10\\\"\\u003e10\\u003c/a\\u003e, \\u003ca href=\\\"#_ENREF_40\\\"\\u003e40\\u003c/a\\u003e, \\u003ca href=\\\"#_ENREF_41\\\"\\u003e41\\u003c/a\\u003e]. We also explored the potential for cross-amplification of the \\u003cem\\u003eT. septentrionalis\\u003c/em\\u003e markers in two closely related species: \\u003cem\\u003eT. arizonensis \\u003c/em\\u003eand \\u003cem\\u003eT. pomonae\\u003c/em\\u003e. These three species belong to \\u003cem\\u003eTrachymyrmex sensu stricto\\u003c/em\\u003e, unlike \\u003cem\\u003eM. turrifex\\u003c/em\\u003e which belongs to a new genus, \\u003cem\\u003eMycetomoellerius\\u003c/em\\u003e [\\u003ca href=\\\"#_ENREF_3\\\"\\u003e3\\u003c/a\\u003e, \\u003ca href=\\\"#_ENREF_33\\\"\\u003e33\\u003c/a\\u003e, \\u003ca href=\\\"#_ENREF_42\\\"\\u003e42\\u003c/a\\u003e]. These markers will provide a new tool to study patterns of attine intraspecific genetic variation and phylogeographic structure.\\u003c/p\\u003e\"},{\"header\":\"Methods\",\"content\":\"\\u003cp\\u003ePrimer sets were developed from a single \\u003cem\\u003eT. septentrionalis\\u003c/em\\u003e forager collected in north Florida ([\\u003ca href=\\\"#_ENREF_10\\\"\\u003e10\\u003c/a\\u003e] Site C; 30.2796, -84.9004) and a single \\u003cem\\u003eM. turrifex\\u003c/em\\u003e forager collected in central Texas (University of Texas at Austin; 30.2861, -97.7297). Illumina sequencing and primer design were conducted at the Savannah River Ecology Laboratory (University of Georgia, USA) in the Molecular Ecology Laboratory. A Covaris S220 Focused-ultrasonicator sheared 1 \\u0026mu;g of genomic DNA to prepare an Illumina paired-end shotgun library following standard Illumina TruSeq DNA Library Kit protocols and using a multiplex identifier adaptor index. Sequencing was conducted on an Illumina HiSeq 3000 platform (San Diego, CA, USA) producing 100-bp paired-end reads. PAL_FINDER v.0.02.03 [\\u003ca href=\\\"#_ENREF_43\\\"\\u003e43\\u003c/a\\u003e] was used to analyze five million resulting reads, and those with di-, tri-, tetra-, penta-, and hexanucleotide microsatellites were extracted. Positive reads were batched to a local installation of Primer3 v.2.0.0 [\\u003ca href=\\\"#_ENREF_44\\\"\\u003e44\\u003c/a\\u003e, \\u003ca href=\\\"#_ENREF_45\\\"\\u003e45\\u003c/a\\u003e] for primer design. Markers for which the primer sequence only occurred three or four times in the five million reads were selected to avoid duplication. From this selection of 9,293 (\\u003cem\\u003eT. septentrionalis\\u003c/em\\u003e) and 6,943 (\\u003cem\\u003eM. turrifex\\u003c/em\\u003e) markers, we chose a subset to test (46 for \\u003cem\\u003eT. septentrionalis\\u003c/em\\u003e; 20 for \\u003cem\\u003eM. turrifex\\u003c/em\\u003e) following an M13-tail PCR protocol [\\u003ca href=\\\"#_ENREF_46\\\"\\u003e46\\u003c/a\\u003e].\\u003c/p\\u003e\\n\\u003cp\\u003eWe extracted genomic DNA from individual worker ants (from restricted portions of their respective ranges; Additional file 1) preserved in 95% ethanol at -20\\u0026deg;C using a QIAamp DNA Micro Kit (QIAGEN). In particular, ants used in the development of \\u003cem\\u003eT. septentrionalis\\u003c/em\\u003e markers were all collected in Florida (i.e., not Texas) (Table 1). For all species, the 10 \\u0026mu;L PCR mix contained 1 \\u0026mu;L of 10X PCR buffer (1.0X; Applied Biosystems), 1 \\u0026mu;L Bioline\\u0026reg; dNTP mix (1 mM each; 0.1 mM proportional to total), 1.5 \\u0026mu;L of 25 mM MgCl\\u003csub\\u003e2\\u003c/sub\\u003e (3.75 mM; Applied Biosystems), 0.5 \\u0026mu;L of 20 \\u0026mu;M BSA (1 \\u0026mu;M; New England Biolabs), 0.3 \\u0026mu;L of 2 \\u0026mu;M tag-labeled primer (0.06 \\u0026mu;M; forward primer with M13-tail), 0.6 \\u0026mu;L of a 2 \\u0026mu;M universal dye-labeled primer (0.12 \\u0026mu;M; FAM label with M13-tail), 1 \\u0026mu;L of 2 \\u0026mu;M unlabeled primer (0.2 \\u0026mu;M; reverse primer), 0.1 \\u0026mu;L of \\u003cem\\u003eTaq \\u003c/em\\u003epolymerase (0.5 U; Applied Biosystems), and 1 \\u0026mu;L of DNA template. PCR cycling conditions consisted of an initial denaturation of 4 min at 95\\u0026deg;C, 25 (or 30) cycles of 30 s at 95\\u0026deg;C, 45 s at the primer-specific annealing temperature identified by a temperature gradient program, 45 s at 72\\u0026deg;C, then 8 cycles of 30 s at 95\\u0026deg;C, 45s at 53\\u0026deg;C, and 45 s at 72\\u0026deg;C, and a final extension of 5 min at 72\\u0026deg;C. PCR products were run on an Applied Biosystems 3730 Genetic Analyzer and fragments were sized with LIZ600 size standard at the University of Texas at Austin DNA Sequencing Facility.\\u003c/p\\u003e\\n\\u003cp\\u003eWe scored alleles in Geneious v.10.2.3 [\\u003ca href=\\\"#_ENREF_47\\\"\\u003e47\\u003c/a\\u003e]. For markers that resulted in high quality PCR product and exhibited polymorphism, we measured genetic variability for each species. We estimated the number of alleles per locus (\\u003cem\\u003eK\\u003c/em\\u003e), observed and expected heterozygosity (H\\u003csub\\u003eo\\u003c/sub\\u003e and H\\u003csub\\u003ee\\u003c/sub\\u003e), and the probability of identity (PI; the probability of two independent samples having the same genotype) using GenAlEx v.6.5 [\\u003ca href=\\\"#_ENREF_48\\\"\\u003e48\\u003c/a\\u003e, \\u003ca href=\\\"#_ENREF_49\\\"\\u003e49\\u003c/a\\u003e]. Sample sizes (numbers of individuals tested) for each marker are provided (Tables 1 and 2, Additional files 2 and 3). We assessed deviations from Hardy-Weinberg equilibrium (HWE) and tested for linkage disequilibrium (LD) across all pairs of markers using GENEPOP v.4.2 [\\u003ca href=\\\"#_ENREF_50\\\"\\u003e50\\u003c/a\\u003e] employing Bonferroni corrections for multiple comparisons. The presence of null alleles was assessed using Microchecker [\\u003ca href=\\\"#_ENREF_51\\\"\\u003e51\\u003c/a\\u003e].\\u003c/p\\u003e\"},{\"header\":\"Results\",\"content\":\"\\u003cp\\u003eFor \\u003cem\\u003eTrachymyrmex septentrionalis \\u003c/em\\u003emarkers tested on \\u003cem\\u003eT. septentrionalis\\u003c/em\\u003e ants, 37 of the original 46 positively amplified with 17 being reliably amplified and scored. The other 20 either showed weak amplification, weak polymorphism (2-4 alleles per marker), monomorphism, or multi-peak profiles. The number of alleles across all samples of the 17 reliable markers ranged from 9 to 33 (average = 16.2). H\\u003csub\\u003eo\\u003c/sub\\u003e varied from 0.11 to 0.89 (average = 0.62), while H\\u003csub\\u003ee\\u003c/sub\\u003e ranged from 0.65 to 0.94 (average = 0.84). After Bonferroni correction, 9 markers showed significant deviations from HWE. There were two cases of LD detected across the 136 paired markers (between Ts4 and Ts5, and Ts3 and Ts43; Table 1; Additional file 4). Most of the loci tested significantly for the presence of null alleles, which suggests that \\u003cem\\u003eT. septentrionalis \\u003c/em\\u003emay have even higher allelic diversity than what we captured in our sampling (Table 1).\\u003c/p\\u003e\\n\\u003cp\\u003eOf the 20 original \\u003cem\\u003eMycetomoellerius turrifex \\u003c/em\\u003emarkers, 17 positively amplified with 12 reliably amplified and scored. The number of alleles across all samples ranged from 4 to 12 (average = 8.9). H\\u003csub\\u003eo\\u003c/sub\\u003e varied from 0.2 to 0.71 (average = 0.49), while H\\u003csub\\u003ee\\u003c/sub\\u003e ranged from 0.58 to 0.88 (average = 0.78). After Bonferroni correction, 6 markers showed significant deviations from HWE. There were no cases of LD detected across the 66 paired markers (Table 2; Additional file 4). In contrast to \\u003cem\\u003eT. septentrionalis\\u003c/em\\u003e, the frequency of null alleles was lower in \\u003cem\\u003eM. turrifex\\u003c/em\\u003e (Table 2).\\u003c/p\\u003e\\n\\u003cp\\u003eWe tested cross-species amplification of the 46 \\u003cem\\u003eT. septentrionalis \\u003c/em\\u003emarkers developed here in both \\u003cem\\u003eT. arizonensis \\u003c/em\\u003eand \\u003cem\\u003eT. pomonae\\u003c/em\\u003e. For \\u003cem\\u003eT. arizonensis \\u003c/em\\u003e(n = 8), 24 markers were positively amplified with 10 reliably scored. Of those 10, all revealed at least 2 alleles, 8 revealed 3 or more alleles, and 9 exhibited some heterozygosity. There were 9 markers in HWE and one violated HWE (Ts38). There was no LD (Additional file 2; Additional file 4). For \\u003cem\\u003eT. pomonae \\u003c/em\\u003e(n = 6), 23 markers were successfully amplified and 12 were reliably scored. Of those 12, all revealed at least 2 alleles, 8 revealed 3 or more alleles, and 10 exhibited some heterozygosity. There were 10 in HWE, one violated HWE (also Ts38), and one could not be computed (Ts7) because there was only one copy of the second allele. There was no LD (Additional file 3; Additional file 4). Only one of the loci in \\u003cem\\u003eT. pomonae\\u003c/em\\u003e and only two in \\u003cem\\u003eT. arizonensis\\u003c/em\\u003e appeared to have null alleles; this likely results from examining ants collected across a few hectares of pine oak forest (Southwest Research Station) (Additional Files 2 and 3).\\u003c/p\\u003e\"},{\"header\":\"Discussion\",\"content\":\"\\u003cp\\u003eThese newly developed microsatellite markers provide a promising new tool for population genetic, mating, dispersal, and phylogeographic studies of \\u003cem\\u003eTrachymyrmex septentrionalis \\u003c/em\\u003eand \\u003cem\\u003eMycetomoellerius turrifex\\u003c/em\\u003e populations across their ranges. Positive cross-species amplification also indicates that some of the \\u003cem\\u003eT. septentrionalis \\u003c/em\\u003emarkers appear to be conserved in at least two other congeneric species (\\u003cem\\u003eT. arizonensis \\u003c/em\\u003eand \\u003cem\\u003eT. pomonae\\u003c/em\\u003e). This indicates that future analyses for additional individuals across a wider geographic region may be informative for understanding similar questions for \\u003cem\\u003eT. arizonensis \\u003c/em\\u003eand \\u003cem\\u003eT. pomonae\\u003c/em\\u003e and perhaps other closely related \\u003cem\\u003eTrachymyrmex\\u003c/em\\u003e. It also seems likely that the markers developed here for \\u003cem\\u003eM. turrifex\\u003c/em\\u003e should cross amplify in closely related \\u003cem\\u003eMycetomoellerius\\u003c/em\\u003e species, such as the highly studied \\u003cem\\u003eM. zeteki\\u003c/em\\u003e [\\u003ca href=\\\"#_ENREF_42\\\"\\u003e42\\u003c/a\\u003e, \\u003ca href=\\\"#_ENREF_52\\\"\\u003e52\\u003c/a\\u003e, \\u003ca href=\\\"#_ENREF_53\\\"\\u003e53\\u003c/a\\u003e], among others.\\u003c/p\\u003e\\n\\u003cp\\u003eWhile all markers across all four species showed polymorphism/allelic variation, several markers (particularly within \\u003cem\\u003eT. septentrionalis\\u003c/em\\u003e) were hypervariable (over 20 alleles per locus; Table 1). These hypervariable markers, collectively with other polymorphic markers, may be informative for investigating intraspecific dynamics at an especially fine-scale (e.g., mating habits such as multiple-mating or multiple-queen hypotheses by comparing relatedness of individuals within colonies). Despite the extensive polymorphism illustrated by these markers, evidence showing the presence of null alleles suggests that more alleles may remain to be discovered. The frequency of null alleles may be more pronounced in populations that already demonstrate significant genetic structure [\\u003ca href=\\\"#_ENREF_54\\\"\\u003e54\\u003c/a\\u003e].\\u003c/p\\u003e\\n\\u003cp\\u003eThe new markers developed here may also be used in conjunction with mtDNA to better understand dispersal, signatures of population expansion or contraction, and phylogeographic history. For example, male and female ants of several species may mediate gene flow differentially through sex-specific dispersal patterns [\\u003ca href=\\\"#_ENREF_13\\\"\\u003e13\\u003c/a\\u003e, \\u003ca href=\\\"#_ENREF_14\\\"\\u003e14\\u003c/a\\u003e, \\u003ca href=\\\"#_ENREF_55\\\"\\u003e55-57\\u003c/a\\u003e], but this has yet been tested in \\u003cem\\u003eTrachymyrmex\\u003c/em\\u003e or \\u003cem\\u003eMycetomoellerius \\u003c/em\\u003eants. By employing marker systems with different modes of inheritance, we may be able to discern how ecological dispersal patterns influence ant genetic structure across populations.\\u003c/p\\u003e\\n\\u003cp\\u003eIn a phylogeographic context, the application of both maternally and biparentally inherited markers may also help to uncover and track the progression of previously unrecognized or poorly understood historical processes such as species origin, divergence, colonization, and genetic drift. For example, the extensive evidence for null alleles in \\u003cem\\u003eT. septentrionalis\\u003c/em\\u003e is likely driven by phylogeographic structure in this species across the relatively restricted range examined in this study (northern Florida) [\\u003ca href=\\\"#_ENREF_10\\\"\\u003e10\\u003c/a\\u003e]. We explored the 17 \\u003cem\\u003eT. septentrionalis\\u003c/em\\u003e markers across a small subset (total n = 12) of \\u003cem\\u003eT. septentrionalis\\u003c/em\\u003e ants from Texas as compared to a random subset of \\u003cem\\u003eT. septentrionalis\\u003c/em\\u003e ants from Florida. The allelic diversity appears to be much higher in Florida than in Texas (see Additional file 5), which may suggest some fundamental genomic differences in populations east and west of the Mississippi River. These preliminary results align with previous mtDNA results that suggest 1) a significant divergence between populations on either side of the Mississippi River and 2) limited genetic diversity west of the Mississippi River [\\u003ca href=\\\"#_ENREF_10\\\"\\u003e10\\u003c/a\\u003e, \\u003ca href=\\\"#_ENREF_11\\\"\\u003e11\\u003c/a\\u003e]. Future analyses comparing the allelic diversity of populations east and west of the Mississippi River would be insightful for better understanding these patterns.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eLimitations\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cul\\u003e\\n\\u003cli\\u003eThe restricted sample size and geographic distribution for \\u003cem\\u003e arizonensis\\u003c/em\\u003e and \\u003cem\\u003eT. pomonae\\u003c/em\\u003e may underestimate potential polymorphic markers in these species.\\u003c/li\\u003e\\n\\u003cli\\u003eMarkers not deviating from HWE in our initial screenings could potentially deviate in other populations, which may reduce the number of usable markers in certain genetic studies; on the other hand, markers deviating from HWE in the studied populations might not deviate in other populations, which would increase the number of markers.\\u003c/li\\u003e\\n\\u003cli\\u003eThe presence of null alleles could be a result of potential phylogeographic structure in the populations studied (especially \\u003cem\\u003e septentrionalis\\u003c/em\\u003e), as a result polymorphism may be underestimated.\\u003c/li\\u003e\\n\\u003cli\\u003eSample sizes per marker for the Texas \\u003cem\\u003e septentrionalis\\u003c/em\\u003e subset were smaller compared to Florida, which may limit our interpretations; however, reduced microsatellite diversity would be consistent with lower mtDNA diversity in western populations (Texas).\\u003c/li\\u003e\\n\\u003c/ul\\u003e\"},{\"header\":\"Abbreviations\",\"content\":\"\\u003cp\\u003ebp: base pair; H\\u003csub\\u003ee\\u003c/sub\\u003e: expected heterozygosity; H\\u003csub\\u003eo\\u003c/sub\\u003e: observed heterozygosity; HWE: Hardy-Weinberg equilibrium; K: number of alleles; LD: linkage disequilibrium; mtDNA: mitochondrial DNA; NGS: next-generation sequencing; PCR: polymerase chain reaction; PI: probability of identity; SNPs: single-nucleotide polymorphisms; T\\u003csub\\u003em\\u003c/sub\\u003e: annealing temperature\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eAcknowledgements\\u003cbr /\\u003e \\u003c/strong\\u003eLarry Gilbert, Steven Gibson and Rob Plowes facilitated collections at the University of Texas at Austin Stengl Biological Station and Brackenridge Field Laboratory. Joshua King and Andrea Lucky provided \\u003cem\\u003eT. septentrionalis\\u003c/em\\u003e specimens from the Orlando and Gainesville, Florida areas, respectively. We thank Geoff Bender, Michele Lanan and Southwestern Research Station staff for providing logistical support and facilitating collection permits in the Coronado National Forest. This is paper No. 3 of the Ant Symbiosis Laboratory.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAuthors\\u0026rsquo; contributions\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eJNS and KK conceived the study, secured funding and permits, and collected samples. AEM, CR, and CS performed DNA extractions, PCR work, and tested/optimized primer pairs on all species. AEM scored alleles and conducted data analysis. AEM and JNS led manuscript writing, and all authors read, edited, and approved the final manuscript.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAvailability of data and material\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe datasets analyzed during the current study are not presently public due to their use in ongoing publication, but the genotypic data for each sample are available upon reasonable request to the corresponding author.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eCompeting interests\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe authors declare they have no competing interests.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eConsent for publication\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eNot applicable.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eEthics approval and consent to participate\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eNot applicable. The current research did not involve human subjects, human material, or human data or animals.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eFunding\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe work was supported primarily by a National Science Foundation CAREER award to JNS (IOS-152822) and DEB-\\u003cstrong\\u003e1354629\\u0026nbsp;to\\u003c/strong\\u003e KK. Additional funding was provided by the UT Tyler Office of Sponsored Research to JNS and Texas Ecolab (www.texasecolab.org), who also provided access to private properties.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\n\\u003cli\\u003eH\\u0026ouml;lldobler B, Wilson EO: \\u003cstrong\\u003eThe Leafcutter Ants\\u003c/strong\\u003e. New York: W.W. Norton; 2011.\\u003c/li\\u003e\\n\\u003cli\\u003eJe\\u0026scaron;ovnik A, Sosa-Calvo J, Lloyd MW, Branstetter MG, Fern\\u0026aacute;ndez F, Schultz TR: \\u003cstrong\\u003ePhylogenomic species delimitation and host-symbiont coevolution in the fungus-farming ant genus \\u003cem\\u003eSericomyrmex \\u003c/em\\u003eMayr (Hymenoptera: Formicidae): ultraconserved elements (UCEs) resolve a recent radiation\\u003c/strong\\u003e. \\u003cem\\u003eSystematic Entomology \\u003c/em\\u003e2017, \\u003cstrong\\u003e42\\u003c/strong\\u003e(3):523-542.\\u003c/li\\u003e\\n\\u003cli\\u003eSchultz TR, Brady SG: \\u003cstrong\\u003eMajor evolutionary transitions in ant agriculture\\u003c/strong\\u003e. \\u003cem\\u003eProceedings of the National Academy of Science, USA \\u003c/em\\u003e2008, \\u003cstrong\\u003e105\\u003c/strong\\u003e(14):5435-5440.\\u003c/li\\u003e\\n\\u003cli\\u003eSchultz TR, Sosa-Calvo J, Brady SG, Lopes CT, Mueller UG, Bacci M, Jr., Vasconcelos HL: \\u003cstrong\\u003eThe most relictual fungus-farming ant species cultivates the most recently evolved and highly domesticated fungal symbiont species\\u003c/strong\\u003e. \\u003cem\\u003eAmerican Naturalist \\u003c/em\\u003e2015, \\u003cstrong\\u003e185\\u003c/strong\\u003e(5):693-703.\\u003c/li\\u003e\\n\\u003cli\\u003eSosa-Calvo J, Schultz TR, Brand\\u0026atilde;o CRF, Klingenberg C, Feitosa RM, Rabeling C, Bacci M, Jr., Lopes CT, Vasconcelos HL: \\u003cstrong\\u003eCyatta abscondita: Taxonomy, Evolution, and Natural History of a New Fungus-Farming Ant Genus from Brazil\\u003c/strong\\u003e. \\u003cem\\u003ePLoS ONE \\u003c/em\\u003e2013, \\u003cstrong\\u003e8\\u003c/strong\\u003e(11):e80498.\\u003c/li\\u003e\\n\\u003cli\\u003eLuiso J, Kellner K, Matthews AE, Mueller UG, Seal JN: \\u003cstrong\\u003eHigh Diversity and Multiple Invasions to North America by fungi grown by the northern most \\u003cem\\u003eTrachymyrmex \\u003c/em\\u003eand \\u003cem\\u003eMycetomoellerius \\u003c/em\\u003eant 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Fern\\u0026aacute;ndez-Mar\\u0026iacute;n H, Ishak HD, Sen R, Linksvayer TA, Mueller UG: \\u003cstrong\\u003eCo-evolutionary patterns and diversification of ant-fungus associations in the asexual fungus-farming ant \\u003cem\\u003eMycocepurus smithii\\u003c/em\\u003e in Panama.\\u003c/strong\\u003e \\u003cem\\u003eJournal of Evolutionary Biology \\u003c/em\\u003e2013, \\u003cstrong\\u003e26\\u003c/strong\\u003e(6):1353-1362.\\u003c/li\\u003e\\n\\u003cli\\u003eRabeling C, Gonzales O, Schultz T, Bacci M, Garcia M, Verhaagh M, Ishak H, Mueller U: \\u003cstrong\\u003eCryptic sexual populations account for genetic diversity and ecological success in a widely distributed, asexual fungus-growing ant.\\u003c/strong\\u003e \\u003cem\\u003eProceedings of the National Academy of Science, USA \\u003c/em\\u003e2011, \\u003cstrong\\u003e108\\u003c/strong\\u003e(30):12366\\u0026ndash;12371.\\u003c/li\\u003e\\n\\u003cli\\u003eKellner K, Seal J, Heinze J: \\u003cstrong\\u003eSex at the margins: 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1.\\u003c/strong\\u003e \\u003cem\\u003eTrachymyrmex septentrionalis\\u003c/em\\u003e polymorphic microsatellite markers analyzed for \\u003cem\\u003eTrachymyrmex septentrionalis\\u003c/em\\u003e ants.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n\\u003ctable border=\\\"1\\\" cellpadding=\\\"0\\\" cellspacing=\\\"0\\\" style=\\\"width:635.3pt;border-collapse:collapse;border:none;\\\" width=\\\"847\\\"\\u003e\\n \\u003ctbody\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd style=\\\"width: 47.65pt;border-top: 1pt solid black;border-left: none;border-bottom: 1pt solid black;border-right: none;padding: 0in 5.4pt;height: 15pt;vertical-align: top;\\\" valign=\\\"top\\\" width=\\\"7.547169811320755%\\\"\\u003e\\n \\u003cp style=\\\"text-align:center;\\\"\\u003e\\u003cstrong\\u003e\\u003cspan style=\\\"color:black;\\\"\\u003eMarker\\u003c/span\\u003e\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 235.85pt;border-top: 1pt solid black;border-left: none;border-bottom: 1pt solid black;border-right: none;padding: 0in 5.4pt;height: 15pt;vertical-align: top;\\\" valign=\\\"top\\\" width=\\\"37.028301886792455%\\\"\\u003e\\n \\u003cp style=\\\"text-align:center;\\\"\\u003e\\u003cstrong\\u003e\\u003cspan style=\\\"color:black;\\\"\\u003ePrimer sequence 5' -\\u0026gt; 3'\\u003c/span\\u003e\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 58.5pt;border-top: 1pt solid black;border-left: none;border-bottom: 1pt solid black;border-right: none;padding: 0in 5.4pt;height: 15pt;vertical-align: top;\\\" valign=\\\"top\\\" width=\\\"9.19811320754717%\\\"\\u003e\\n \\u003cp style=\\\"text-align:center;\\\"\\u003e\\u003cstrong\\u003e\\u003cspan style=\\\"color:black;\\\"\\u003eRepeat motif\\u003c/span\\u003e\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 47.65pt;border-top: 1pt solid black;border-left: none;border-bottom: 1pt solid black;border-right: 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235.85pt;border: none;padding: 0in 5.4pt;height: 15pt;vertical-align: top;\\\" valign=\\\"top\\\" width=\\\"37.028301886792455%\\\"\\u003e\\n \\u003cp\\u003e\\u003cspan style=\\\"color:black;\\\"\\u003eF: CGTGTCAAGTATGTTCCCGC\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 58.5pt;border: none;padding: 0in 5.4pt;height: 15pt;vertical-align: top;\\\" valign=\\\"top\\\" width=\\\"9.19811320754717%\\\"\\u003e\\n \\u003cp style=\\\"text-align:center;\\\"\\u003e\\u003cspan style=\\\"color:black;\\\"\\u003eTGCG\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 47.65pt;border: none;padding: 0in 5.4pt;height: 15pt;vertical-align: top;\\\" valign=\\\"top\\\" width=\\\"7.547169811320755%\\\"\\u003e\\n \\u003cp style=\\\"text-align:center;\\\"\\u003e\\u003cspan style=\\\"color:black;\\\"\\u003e61.3\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 51.35pt;border: none;padding: 0in 5.4pt;height: 15pt;vertical-align: top;\\\" valign=\\\"top\\\" width=\\\"8.018867924528301%\\\"\\u003e\\n \\u003cp style=\\\"text-align:center;\\\"\\u003e\\u003cspan style=\\\"color:black;\\\"\\u003e174-316\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 0.5in;border: none;padding: 0in 5.4pt;height: 15pt;vertical-align: top;\\\" valign=\\\"top\\\" width=\\\"5.660377358490566%\\\"\\u003e\\n \\u003cp style=\\\"text-align:center;\\\"\\u003e\\u003cspan style=\\\"color:black;\\\"\\u003e48\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 21.8pt;border: none;padding: 0in 5.4pt;height: 15pt;vertical-align: top;\\\" valign=\\\"top\\\" width=\\\"3.419811320754717%\\\"\\u003e\\n \\u003cp style=\\\"text-align:center;\\\"\\u003e\\u003cspan style=\\\"color:black;\\\"\\u003e28\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 41.2pt;border: none;padding: 0in 5.4pt;height: 15pt;vertical-align: top;\\\" valign=\\\"top\\\" width=\\\"6.485849056603773%\\\"\\u003e\\n \\u003cp style=\\\"text-align:center;\\\"\\u003e\\u003cspan style=\\\"color:black;\\\"\\u003e0.60\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 47.65pt;border: none;padding: 0in 5.4pt;height: 15pt;vertical-align: top;\\\" valign=\\\"top\\\" width=\\\"7.547169811320755%\\\"\\u003e\\n \\u003cp style=\\\"text-align:center;\\\"\\u003e\\u003cspan style=\\\"color:black;\\\"\\u003e0.89\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 47.65pt;border: none;padding: 0in 5.4pt;height: 15pt;vertical-align: top;\\\" valign=\\\"top\\\" width=\\\"7.547169811320755%\\\"\\u003e\\n \\u003cp style=\\\"text-align:center;\\\"\\u003e\\u003cspan style=\\\"color:black;\\\"\\u003e0.02\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd style=\\\"width: 47.65pt;border: none;padding: 0in 5.4pt;height: 15pt;vertical-align: top;\\\" valign=\\\"top\\\" width=\\\"7.547169811320755%\\\"\\u003e\\n \\u003cbr\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"3\\\" style=\\\"width: 4.75in;border: none;padding: 0in 5.4pt;height: 15pt;vertical-align: top;\\\" valign=\\\"top\\\" width=\\\"53.77358490566038%\\\"\\u003e\\n \\u003cp\\u003e\\u003cspan style=\\\"color:black;\\\"\\u003eR: AGTTTCAGGCGCAGGTAGC\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 51.35pt;border: none;padding: 0in 5.4pt;height: 15pt;vertical-align: top;\\\" valign=\\\"top\\\" width=\\\"8.018867924528301%\\\"\\u003e\\n \\u003cbr\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 0.5in;border: none;padding: 0in 5.4pt;height: 15pt;vertical-align: top;\\\" valign=\\\"top\\\" width=\\\"5.660377358490566%\\\"\\u003e\\n \\u003cbr\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 21.8pt;border: none;padding: 0in 5.4pt;height: 15pt;vertical-align: top;\\\" valign=\\\"top\\\" width=\\\"3.419811320754717%\\\"\\u003e\\n \\u003cbr\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 41.2pt;border: none;padding: 0in 5.4pt;height: 15pt;vertical-align: top;\\\" valign=\\\"top\\\" width=\\\"6.485849056603773%\\\"\\u003e\\n \\u003cbr\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 47.65pt;border: none;padding: 0in 5.4pt;height: 15pt;vertical-align: top;\\\" valign=\\\"top\\\" width=\\\"7.547169811320755%\\\"\\u003e\\n \\u003cbr\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 47.65pt;border: none;padding: 0in 5.4pt;height: 15pt;vertical-align: top;\\\" valign=\\\"top\\\" width=\\\"7.547169811320755%\\\"\\u003e\\n \\u003cbr\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd style=\\\"width: 47.65pt;border: none;padding: 0in 5.4pt;height: 15pt;vertical-align: top;\\\" valign=\\\"top\\\" width=\\\"7.547169811320755%\\\"\\u003e\\n \\u003cp\\u003e\\u003cspan style=\\\"color:black;\\\"\\u003eTs46 \\u003csup\\u003ec\\u003c/sup\\u003e\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 235.85pt;border: none;padding: 0in 5.4pt;height: 15pt;vertical-align: top;\\\" valign=\\\"top\\\" width=\\\"37.028301886792455%\\\"\\u003e\\n \\u003cp\\u003e\\u003cspan style=\\\"color:black;\\\"\\u003eF: GTACGCACATCGTGCTAAACG\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 58.5pt;border: none;padding: 0in 5.4pt;height: 15pt;vertical-align: top;\\\" valign=\\\"top\\\" width=\\\"9.19811320754717%\\\"\\u003e\\n \\u003cp style=\\\"text-align:center;\\\"\\u003e\\u003cspan style=\\\"color:black;\\\"\\u003eACCG\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 47.65pt;border: none;padding: 0in 5.4pt;height: 15pt;vertical-align: top;\\\" valign=\\\"top\\\" width=\\\"7.547169811320755%\\\"\\u003e\\n \\u003cp style=\\\"text-align:center;\\\"\\u003e\\u003cspan style=\\\"color:black;\\\"\\u003e60.5\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 51.35pt;border: none;padding: 0in 5.4pt;height: 15pt;vertical-align: top;\\\" valign=\\\"top\\\" width=\\\"8.018867924528301%\\\"\\u003e\\n \\u003cp style=\\\"text-align:center;\\\"\\u003e\\u003cspan style=\\\"color:black;\\\"\\u003e310-348\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 0.5in;border: none;padding: 0in 5.4pt;height: 15pt;vertical-align: top;\\\" valign=\\\"top\\\" width=\\\"5.660377358490566%\\\"\\u003e\\n \\u003cp style=\\\"text-align:center;\\\"\\u003e\\u003cspan style=\\\"color:black;\\\"\\u003e46\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 21.8pt;border: none;padding: 0in 5.4pt;height: 15pt;vertical-align: top;\\\" valign=\\\"top\\\" width=\\\"3.419811320754717%\\\"\\u003e\\n \\u003cp style=\\\"text-align:center;\\\"\\u003e\\u003cspan style=\\\"color:black;\\\"\\u003e20\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 41.2pt;border: none;padding: 0in 5.4pt;height: 15pt;vertical-align: top;\\\" valign=\\\"top\\\" width=\\\"6.485849056603773%\\\"\\u003e\\n \\u003cp style=\\\"text-align:center;\\\"\\u003e\\u003cspan style=\\\"color:black;\\\"\\u003e0.78\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 47.65pt;border: none;padding: 0in 5.4pt;height: 15pt;vertical-align: top;\\\" valign=\\\"top\\\" width=\\\"7.547169811320755%\\\"\\u003e\\n \\u003cp style=\\\"text-align:center;\\\"\\u003e\\u003cspan style=\\\"color:black;\\\"\\u003e0.92\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 47.65pt;border: none;padding: 0in 5.4pt;height: 15pt;vertical-align: top;\\\" valign=\\\"top\\\" width=\\\"7.547169811320755%\\\"\\u003e\\n \\u003cp style=\\\"text-align:center;\\\"\\u003e\\u003cspan style=\\\"color:black;\\\"\\u003e0.01\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd style=\\\"width: 47.65pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid black;padding: 0in 5.4pt;height: 15pt;vertical-align: top;\\\" valign=\\\"top\\\" width=\\\"7.547169811320755%\\\"\\u003e\\n \\u003cbr\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"3\\\" style=\\\"width: 4.75in;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid black;padding: 0in 5.4pt;height: 15pt;vertical-align: top;\\\" valign=\\\"top\\\" width=\\\"53.77358490566038%\\\"\\u003e\\n \\u003cp\\u003e\\u003cspan style=\\\"color:black;\\\"\\u003eR: AGCGGTGGTGGTTTCACG\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 51.35pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid black;padding: 0in 5.4pt;height: 15pt;vertical-align: top;\\\" valign=\\\"top\\\" width=\\\"8.018867924528301%\\\"\\u003e\\n \\u003cbr\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 0.5in;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid black;padding: 0in 5.4pt;height: 15pt;vertical-align: top;\\\" valign=\\\"top\\\" width=\\\"5.660377358490566%\\\"\\u003e\\n \\u003cbr\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 21.8pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid black;padding: 0in 5.4pt;height: 15pt;vertical-align: top;\\\" valign=\\\"top\\\" width=\\\"3.419811320754717%\\\"\\u003e\\n \\u003cbr\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 41.2pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid black;padding: 0in 5.4pt;height: 15pt;vertical-align: top;\\\" valign=\\\"top\\\" width=\\\"6.485849056603773%\\\"\\u003e\\n \\u003cbr\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 47.65pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid black;padding: 0in 5.4pt;height: 15pt;vertical-align: top;\\\" valign=\\\"top\\\" width=\\\"7.547169811320755%\\\"\\u003e\\n \\u003cbr\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 47.65pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid black;padding: 0in 5.4pt;height: 15pt;vertical-align: top;\\\" valign=\\\"top\\\" width=\\\"7.547169811320755%\\\"\\u003e\\n \\u003cbr\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/tbody\\u003e\\n\\u003c/table\\u003e\\n\\u003cp style=\\\"line-height:200%;\\\"\\u003eDetails include: marker information, primer sequences, repeat motif, annealing temperature (T\\u003csub\\u003em\\u003c/sub\\u003e), size range of observed alleles given in base pairs (bp), number of individuals genotyped (N), number of alleles observed (K), observed heterozygosity (H\\u003csub\\u003eo\\u003c/sub\\u003e), expected heterozygosity (H\\u003csub\\u003ee\\u003c/sub\\u003e), and probability of identity (PI). Superscript \\u003csup\\u003ea\\u003c/sup\\u003e indicates that 30 cycles were used in the PCR profile, superscript \\u003csup\\u003eb\\u003c/sup\\u003e indicates deviation from Hardy-Weinberg expectations after Bonferroni corrections, and superscript \\u003csup\\u003ec\\u003c/sup\\u003e indicates the marker shows evidence of null alleles.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eTable 2.\\u003c/strong\\u003e \\u003cem\\u003eMycetomoellerius\\u0026nbsp;turrifex\\u003c/em\\u003e polymorphic microsatellite markers analyzed for \\u003cem\\u003eMycetomoellerius\\u0026nbsp;turrifex\\u003c/em\\u003e ants.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n\\u003ctable border=\\\"1\\\" cellpadding=\\\"0\\\" cellspacing=\\\"0\\\" style=\\\"width:9.0in;border-collapse:collapse;border:none;\\\" width=\\\"864\\\"\\u003e\\n \\u003ctbody\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd style=\\\"width: 48pt;border-top: 1pt solid black;border-left: none;border-bottom: 1pt solid black;border-right: none;padding: 0in 5.4pt;height: 15pt;vertical-align: top;\\\" valign=\\\"top\\\" width=\\\"7.407407407407407%\\\"\\u003e\\n \\u003cp style=\\\"text-align:center;\\\"\\u003e\\u003cstrong\\u003e\\u003cspan style=\\\"color:black;\\\"\\u003eMarker\\u003c/span\\u003e\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 244.5pt;border-top: 1pt solid black;border-left: none;border-bottom: 1pt solid black;border-right: none;padding: 0in 5.4pt;height: 15pt;vertical-align: top;\\\" valign=\\\"top\\\" width=\\\"37.73148148148148%\\\"\\u003e\\n \\u003cp style=\\\"text-align:center;\\\"\\u003e\\u003cstrong\\u003e\\u003cspan style=\\\"color:black;\\\"\\u003ePrimer sequence 5' -\\u0026gt; 3'\\u003c/span\\u003e\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 48pt;border-top: 1pt solid black;border-left: none;border-bottom: 1pt solid black;border-right: none;padding: 0in 5.4pt;height: 15pt;vertical-align: top;\\\" valign=\\\"top\\\" width=\\\"7.407407407407407%\\\"\\u003e\\n \\u003cp style=\\\"text-align:center;\\\"\\u003e\\u003cstrong\\u003e\\u003cspan style=\\\"color:black;\\\"\\u003eRepeat motif\\u003c/span\\u003e\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 48pt;border-top: 1pt solid black;border-left: none;border-bottom: 1pt solid black;border-right: none;padding: 0in 5.4pt;height: 15pt;vertical-align: top;\\\" valign=\\\"top\\\" width=\\\"7.407407407407407%\\\"\\u003e\\n \\u003cp style=\\\"text-align:center;\\\"\\u003e\\u003cstrong\\u003e\\u003cspan style=\\\"color:black;\\\"\\u003eT\\u003csub\\u003em\\u003c/sub\\u003e (\\u003c/span\\u003e\\u003cspan style=\\\"color:#545454;background:white;\\\"\\u003e°\\u003c/span\\u003e\\u003cspan style=\\\"color:black;\\\"\\u003eC)\\u003c/span\\u003e\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 48pt;border-top: 1pt solid black;border-left: none;border-bottom: 1pt solid black;border-right: none;padding: 0in 5.4pt;height: 15pt;vertical-align: top;\\\" valign=\\\"top\\\" width=\\\"7.407407407407407%\\\"\\u003e\\n \\u003cp style=\\\"text-align:center;\\\"\\u003e\\u003cstrong\\u003e\\u003cspan style=\\\"color:black;\\\"\\u003eSize (bp)\\u003c/span\\u003e\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 0.5in;border-top: 1pt solid black;border-left: none;border-bottom: 1pt solid black;border-right: none;padding: 0in 5.4pt;height: 15pt;vertical-align: top;\\\" valign=\\\"top\\\" width=\\\"5.555555555555555%\\\"\\u003e\\n \\u003cp style=\\\"text-align:center;\\\"\\u003e\\u003cstrong\\u003e\\u003cspan style=\\\"color:black;\\\"\\u003eN\\u003c/span\\u003e\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 31.5pt;border-top: 1pt solid black;border-left: none;border-bottom: 1pt solid black;border-right: none;padding: 0in 5.4pt;height: 15pt;vertical-align: top;\\\" valign=\\\"top\\\" width=\\\"4.861111111111111%\\\"\\u003e\\n \\u003cp style=\\\"text-align:center;\\\"\\u003e\\u003cstrong\\u003e\\u003cspan style=\\\"color:black;\\\"\\u003eK\\u003c/span\\u003e\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 48pt;border-top: 1pt solid black;border-left: none;border-bottom: 1pt solid black;border-right: none;padding: 0in 5.4pt;height: 15pt;vertical-align: top;\\\" valign=\\\"top\\\" width=\\\"7.407407407407407%\\\"\\u003e\\n \\u003cp style=\\\"text-align:center;\\\"\\u003e\\u003cstrong\\u003e\\u003cspan style=\\\"color:black;\\\"\\u003eH\\u003csub\\u003eo\\u003c/sub\\u003e\\u003c/span\\u003e\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 48pt;border-top: 1pt solid black;border-left: none;border-bottom: 1pt solid black;border-right: none;padding: 0in 5.4pt;height: 15pt;vertical-align: top;\\\" valign=\\\"top\\\" width=\\\"7.407407407407407%\\\"\\u003e\\n \\u003cp style=\\\"text-align:center;\\\"\\u003e\\u003cstrong\\u003e\\u003cspan style=\\\"color:black;\\\"\\u003eH\\u003csub\\u003ee\\u003c/sub\\u003e\\u003c/span\\u003e\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 48pt;border-top: 1pt solid black;border-left: none;border-bottom: 1pt solid black;border-right: none;padding: 0in 5.4pt;height: 15pt;vertical-align: top;\\\" valign=\\\"top\\\" width=\\\"7.407407407407407%\\\"\\u003e\\n \\u003cp style=\\\"text-align:center;\\\"\\u003e\\u003cstrong\\u003e\\u003cspan 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style=\\\"color:black;\\\"\\u003e0.47\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 48pt;border: none;padding: 0in 5.4pt;height: 15pt;vertical-align: top;\\\" valign=\\\"top\\\" width=\\\"7.407407407407407%\\\"\\u003e\\n \\u003cp style=\\\"text-align:center;\\\"\\u003e\\u003cspan style=\\\"color:black;\\\"\\u003e0.88\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 48pt;border: none;padding: 0in 5.4pt;height: 15pt;vertical-align: top;\\\" valign=\\\"top\\\" width=\\\"7.407407407407407%\\\"\\u003e\\n \\u003cp style=\\\"text-align:center;\\\"\\u003e\\u003cspan style=\\\"color:black;\\\"\\u003e0.03\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd style=\\\"width: 48pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid black;padding: 0in 5.4pt;height: 15pt;vertical-align: top;\\\" valign=\\\"top\\\" width=\\\"7.407407407407407%\\\"\\u003e\\n \\u003cbr\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"4\\\" style=\\\"width: 388.5pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid black;padding: 0in 5.4pt;height: 15pt;vertical-align: top;\\\" valign=\\\"top\\\" width=\\\"59.9537037037037%\\\"\\u003e\\n \\u003cp\\u003e\\u003cspan style=\\\"color:black;\\\"\\u003eR: CCCACAGTAATGTCCTAGTAATGTCC\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 0.5in;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid black;padding: 0in 5.4pt;height: 15pt;vertical-align: top;\\\" valign=\\\"top\\\" width=\\\"5.555555555555555%\\\"\\u003e\\n \\u003cbr\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 31.5pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid black;padding: 0in 5.4pt;height: 15pt;vertical-align: top;\\\" valign=\\\"top\\\" width=\\\"4.861111111111111%\\\"\\u003e\\n \\u003cbr\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 48pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid black;padding: 0in 5.4pt;height: 15pt;vertical-align: top;\\\" valign=\\\"top\\\" width=\\\"7.407407407407407%\\\"\\u003e\\n \\u003cbr\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 48pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid black;padding: 0in 5.4pt;height: 15pt;vertical-align: top;\\\" valign=\\\"top\\\" width=\\\"7.407407407407407%\\\"\\u003e\\n \\u003cbr\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 48pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid black;padding: 0in 5.4pt;height: 15pt;vertical-align: top;\\\" valign=\\\"top\\\" width=\\\"7.407407407407407%\\\"\\u003e\\n \\u003cbr\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/tbody\\u003e\\n\\u003c/table\\u003e\\n\\u003cp style=\\\"line-height:200%;\\\"\\u003eDetails include: marker information, primer sequences, repeat motif, annealing temperature (T\\u003csub\\u003em\\u003c/sub\\u003e), size range of observed alleles given in base pairs (bp), number of individuals genotyped (N), number of alleles observed (K), observed heterozygosity (H\\u003csub\\u003eo\\u003c/sub\\u003e), expected heterozygosity (H\\u003csub\\u003ee\\u003c/sub\\u003e), and probability of identity (PI). Superscript \\u003csup\\u003ea\\u003c/sup\\u003e indicates that 30 cycles were used in the PCR profile, superscript \\u003csup\\u003eb\\u003c/sup\\u003e indicates deviation from Hardy-Weinberg expectations after Bonferroni corrections, and superscript \\u003csup\\u003ec\\u003c/sup\\u003e indicates the marker shows evidence of null alleles.\\u003c/p\\u003e\"},{\"header\":\"Additional File Legends\",\"content\":\"\\u003cp\\u003eAdditional file 1 [AdditionalFile1.docx]. List of individual ant samples used in this study. The table includes individual sample name, host species, state of collection, general sampling location, and coordinates of collection location (unless the property was under private ownership).\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eAdditional file 2 [AdditionalFile2.docx]. Details of the 10 \\u003cem\\u003eTrachymyrmex septentrionalis\\u003c/em\\u003e polymorphic microsatellite markers analyzed for cross-amplification in \\u003cem\\u003eTrachymyrmex arizonensis\\u003c/em\\u003e.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eAdditional file 3 [AdditionalFile3.docx]. Details of the 12 \\u003cem\\u003eTrachymyrmex septentrionalis\\u003c/em\\u003e polymorphic microsatellite markers analyzed for cross-amplification in \\u003cem\\u003eTrachymyrmex pomonae\\u003c/em\\u003e.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eAdditional file 4 [AdditionalFile4.docx]. Pairwise comparison results of the linkage disequilibrium analyses for all four species.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eAdditional file 5 [AdditionalFile5.docx]. Details of the 17 \\u003cem\\u003eTrachymyrmex septentrionalis\\u003c/em\\u003e polymorphic microsatellite markers analyzed for \\u003cem\\u003eTrachymyrmex septentrionalis\\u003c/em\\u003e ants across Texas (TX) and Florida (FL), highlighting the differences between two distant populations. Details include: marker name, size range of observed alleles given in base pairs (bp), number of individuals genotyped (N), number of alleles observed (K).\\u003c/p\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":false,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":true,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"bmc-research-notes\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"resn\",\"sideBox\":\"Learn more about [BMC Research Notes](http://bmcresnotes.biomedcentral.com)\",\"snPcode\":\"\",\"submissionUrl\":\"https://www.editorialmanager.com/resn/default.aspx\",\"title\":\"BMC Research Notes\",\"twitterHandle\":\"@BMC_series\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"em\",\"reportingPortfolio\":\"BMC Series\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true},\"keywords\":\"Attini, fungus-gardening ant, intraspecific genetic diversity, microsatellites, population genetics\",\"lastPublishedDoi\":\"10.21203/rs.2.21905/v2\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.2.21905/v2\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"Objective\\nThe objective of this study is to develop and identify polymorphic microsatellite markers for fungus-gardening (attine) ants in the genus Trachymyrmex sensu lato . These ants are important ecosystem engineers and have been a model group for understanding complex symbiotic systems, but very little is understood about the intraspecific genetic patterns across most North American attine species. These microsatellite markers will help to better study intraspecific population genetic structure, gene flow, mating habits, and phylogeographic patterns in these species and potentially other congeners.\\nResults\\nUsing next-generation sequencing techniques, we identified 17 and 12 polymorphic microsatellite markers from T. septentrionalis and Mycetomoellerius (formerly Trachymyrmex ) turrifex , respectively, and assessed the genetic diversity of each marker. We also analyzed the cross-amplification success of the T. septentrionalis markers in two other closely related Trachymyrmex species, and identified 10 and 12 polymorphic markers for T. arizonensis and T. pomonae , respectively.\",\"manuscriptTitle\":\"Development, characterization, and cross-amplification of polymorphic microsatellite markers for North American Trachymyrmex and Mycetomoellerius ants\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":2,\"date\":\"2020-03-09 18:55:59\",\"doi\":\"10.21203/rs.2.21905/v2\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0},{\"type\":\"decision\",\"content\":\"Accept\",\"date\":\"2020-03-10T12:00:00+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorAssigned\",\"content\":\"\",\"date\":\"2020-03-05T12:00:00+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"checksComplete\",\"content\":\"\",\"date\":\"2020-03-04T12:00:00+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorInvited\",\"content\":\"\",\"date\":\"2020-03-04T12:00:00+00:00\",\"index\":\"\",\"fulltext\":\"\"}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"bmc-research-notes\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"resn\",\"sideBox\":\"Learn more about [BMC Research Notes](http://bmcresnotes.biomedcentral.com)\",\"snPcode\":\"\",\"submissionUrl\":\"https://www.editorialmanager.com/resn/default.aspx\",\"title\":\"BMC Research Notes\",\"twitterHandle\":\"@BMC_series\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"em\",\"reportingPortfolio\":\"BMC Series\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true}},{\"code\":1,\"date\":\"2020-01-27 22:02:52\",\"doi\":\"10.21203/rs.2.21905/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0},{\"type\":\"decision\",\"content\":\"Major revision\",\"date\":\"2020-02-14T12:00:00+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2020-02-12T12:00:00+00:00\",\"index\":1,\"fulltext\":\"Recommendation: Reviewer's comments unavailable due to the journal's policy.\\n\"},{\"type\":\"editorAssigned\",\"content\":\"\",\"date\":\"2020-01-22T12:00:00+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"reviewersInvited\",\"content\":\"\",\"date\":\"2020-01-22T12:00:00+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"\",\"date\":\"2020-01-22T12:00:00+00:00\",\"index\":1,\"fulltext\":\"\"},{\"type\":\"submitted\",\"content\":\"\",\"date\":\"2020-01-21T12:00:00+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"checksComplete\",\"content\":\"\",\"date\":\"2020-01-21T12:00:00+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorInvited\",\"content\":\"\",\"date\":\"2020-01-21T12:00:00+00:00\",\"index\":\"\",\"fulltext\":\"\"}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"bmc-research-notes\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"resn\",\"sideBox\":\"Learn more about [BMC Research Notes](http://bmcresnotes.biomedcentral.com)\",\"snPcode\":\"\",\"submissionUrl\":\"https://www.editorialmanager.com/resn/default.aspx\",\"title\":\"BMC Research Notes\",\"twitterHandle\":\"@BMC_series\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"em\",\"reportingPortfolio\":\"BMC Series\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"d80e86db-8a7f-4756-9eb7-ada40caceda7\",\"owner\":[],\"postedDate\":\"March 9th, 2020\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"published-in-journal\",\"subjectAreas\":[{\"id\":53450,\"name\":\"Population Genetics\"}],\"tags\":[],\"updatedAt\":\"2021-07-27T21:11:03+00:00\",\"versionOfRecord\":{\"articleIdentity\":\"rs-12487\",\"link\":\"https://doi.org/10.1186/s13104-020-05015-3\",\"journal\":{\"identity\":\"bmc-research-notes\",\"isVorOnly\":false,\"title\":\"BMC Research Notes\"},\"publishedOn\":\"2020-03-24 21:11:03\",\"publishedOnDateReadable\":\"March 24th, 2020\"},\"versionCreatedAt\":\"2020-03-09 18:55:59\",\"video\":\"\",\"vorDoi\":\"10.1186/s13104-020-05015-3\",\"vorDoiUrl\":\"https://doi.org/10.1186/s13104-020-05015-3\",\"workflowStages\":[]},\"version\":\"v2\",\"identity\":\"rs-12487\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"identity\":\"rs-12487\",\"version\":[\"v2\"]},\"buildId\":\"7rjqhiLT3MXkJMwkYKINL\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}