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Synonymization of the male-based ant genus Phaulomyrma (Hymenoptera, Formicidae) with Leptanilla based upon Bayesian total-evidence phylogenetic inference

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Although molecular data have proven indispensable in confidently resolving the phylogeny of many clades across the tree of life, these data may be inaccessible for certain taxa. The resolution of taxonomy in the ant subfamily Leptanillinae is made problematic by the absence of DNA sequence data for leptanilline taxa that are known only from male specimens, including the monotypic genus Phaulomyrma Wheeler & Wheeler. Focusing upon the considerable diversity of undescribed male leptanilline morphospecies, the phylogeny of 38 putative morphospecies sampled from across the Leptanillinae, plus an outgroup, is inferred from 11 nuclear loci and 41 discrete male morphological characters using a Bayesian total-evidence framework, with Phaulomyrma represented by morphological data only. Based upon the results of this analysis Phaulomyrma is synonymized with Leptanilla Emery, and male-based diagnoses for Leptanilla that are grounded in phylogeny are provided, under both broad and narrow circumscriptions of that genus. This demonstrates the potential utility of a total-evidence approach in inferring the phylogeny of rare extant taxa for which molecular data are unavailable and begins a long-overdue systematic revision of the Leptanillinae that is focused on male material.
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Phylogeny of the Male-Based Ant Genus Phaulomyrma
1
Synonymization of the male-based ant genus Phaulomyrma (Hymenoptera, Formicidae) 1
with Leptanilla based upon Bayesian total-evidence phylogenetic inference 2
Zachary H. Griebenow 3
Abstract 4
Although molecular data have proven indispensable in confidently resolving the phylogeny of 5
many clades across the tree of life, these data may be inaccessible for certain taxa. The resolution 6
of taxonomy in the ant subfamily Leptanillinae is made problematic by the absence of DNA 7
sequence data for leptanilline taxa that are known only from male specimens, including the 8
monotypic genus Phaulomyrma Wheeler & Wheeler. Focusing upon the considerable diversity 9
of undescribed male leptanilline morphospecies, the phylogeny of 38 putative morphospecies 10
sampled from across the Leptanillinae, plus an outgroup, is inferred from 11 nuclear loci and 41 11
discrete male morphological characters using a Bayesian total-evidence framework, with 12
Phaulomyrma represented by morphological data only. Based upon the results of this analysis 13
Phaulomyrma is synonymized with Leptanilla Emery, and male-based diagnoses for Leptanilla 14
that are grounded in phylogeny are provided, under both broad and narrow circumscriptions of 15
that genus. This demonstrates the potential utility of a total-evidence approach in inferring the 16
phylogeny of rare extant taxa for which molecular data are unavailable and begins a long-17
overdue systematic revision of the Leptanillinae that is focused on male material. 18
19
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Phylogeny of the Male-Based Ant Genus Phaulomyrma
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Introduction 20
Over the past three decades, DNA sequences have provided great insight into phylogenetic 21
relationships across the Metazoa, including the insects (Kjer et al. 2018). The application of 22
maximum-likelihood and Bayesian statistical methods to analysis of genetic data has robustly 23
resolved many problems that were intractable when using morphological data alone (e.g. Niehuis 24
et al. 2012; Wipfler et al. 2019). However, DNA sequences may be unavailable for some taxa, 25
necessitating the integration of morphological and molecular data under the same inferential 26
framework. Fossils are the most obvious example of this: these are valuable for calibration of 27
phylogenies in absolute time under a Bayesian approach, preferably with their topological 28
position being inferred from the data (Ronquist et al. 2012; O’Reilly et al. 2015; Bapst et al. 29
2016; Matzke & Wright 2016). Although the inclusion of fossils for the purposes of “tip-dating” 30
has received the bulk of attention in Bayesian total-evidence phylogenetic inference, the lack of 31
molecular data may afflict rare extant taxa as well (Sánchez et al. 2016; Robertson & Moore 32
2016). This is problematic if the affinities of these taxa are not immediately clear from 33
morphology alone. 34
The ant subfamily Leptanillinae (Hymenoptera: Formicidae) is an apt test case for methods to 35
resolve this problem. A group of small, hypogaeic ants largely restricted to the Old World tropics 36
and subtropics, the Leptanillinae are understood to be one of the earliest-diverging lineages in 37
the ant crown-group (Rabeling et al. 2008; Kück et al. 2011; Borowiec et al. 2019; Boudinot et 38
al. submitted). Four leptanilline genera—Scyphodon Brues, 1925; Phaulomyrma Wheeler & 39
Wheeler, 1930; Noonilla Petersen, 1968; and Yavnella Kugler, 1986have been described solely 40
from males, as have many species of Leptanilla Emery, 1870 (cf. Bolton 1990). Recent 41
molecular data indicate that the type species of Yavnella and a specimen provisionally assigned 42
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Phylogeny of the Male-Based Ant Genus Phaulomyrma
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to Phaulomyrma are nested within a clade of putative Leptanilla morphospecies (Borowiec et al. 43
2019). Moreover, although Scyphodon anomalum Brues, 1925 and Noonilla copiosa Petersen, 44
1968 exhibit bizarre autapomorphies such as hypertrophied mandibles (Brues 1925) and a 45
ventromedian genital “trigger” (Petersen 1968), respectively, these ants are otherwise similar to 46
males attributed to Leptanilla (Boudinot 2015). 47
This indicates a need for a systematic revision of the Leptanilline, but almost all published 48
taxonomic study of the group has been descriptive without recourse to molecular phylogeny, 49
with the exceptions being revisions to our concept of the subfamily. Multi-locus DNA datasets 50
demonstrated that the enigmatic Afrotropical genus Apomyrma Gotwald, Brown & Lévieux, 51
1971 is closely related to the Amblyoponinae rather than the Leptanillinae (Brady et al. 2006; 52
Moreau et al. 2006), and that the superficially similar Asian genus Opamyrma Yamane, Bui & 53
Eguchi, 2008 is in fact sister to the remaining Leptanillinae (Ward & Fisher 2016). None of these 54
studies focused upon the Leptanillinae or the internal phylogeny of this clade. Such a study must 55
confront two challenges: first, the lack of DNA sequences for certain critical taxa across the 56
Leptanillinae (e.g., Scyphodon), which hampers any attempt to confidently resolve relationships 57
among these; second, the definition of genera based only upon males, which prevents an 58
integrated phylogenetic classification of the Leptanillinae, since phenotypes of only one sex are 59
considered. 60
The dissociation of leptanilline castes results from collecting bias. Subterranean workers have 61
been largely collected with lavage de terre methodology (López et al. 1994; Wong and Guénard 62
2016), Winkler trapping (Belshaw & Bolton 1994; Leong et al. 2018), and subterranean pitfall 63
traps (Wong & Guénard 2016; Man et al. 2017); whereas male leptanillines are typically 64
collected by sweeping foliage or by deploying Malaise or pan traps (Robertson 2000). None of 65
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Phylogeny of the Male-Based Ant Genus Phaulomyrma
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these methods are likely to collect males in association with workers, nor is the queen caste often 66
collected in association with conspecifics. Contrasting with the alate condition observed in most 67
ants, queens described from the tribe Leptanillini are completely wingless and blind (Emery 68
1870; Kutter 1948; Masuko 1990; López et al. 1994; Ogata et al. 1995), meaning that these are 69
no more likely to be collected than corresponding workers. Queens belonging to other 70
leptanilline lineages (Opamyrma and Anomalomyrmini) are alate so far as is known (Bolton 71
1990; Baroni Urbani & de Andrade 2008; Borowiec et al. 2011; Chen et al. 2017; Hsu et al. 72
2017; Man et al. 2017), save for an apparent record of queen polyphenism in an undescribed 73
Protanilla Taylor, 1990 (Billen et al. 2013), but are infrequently collected. 74
Therefore, the bulk of known leptanilline diversity, most of it undescribed, is represented by 75
exclusively male material. In some cases, molecular data are inaccessible for male morphotaxa 76
due to paucity of suitably recent specimens, obliging a total-evidence approach to infer the 77
phylogeny of these lineages. This study uses such an approach to resolve the position of the 78
male-based species Phaulomyrma javana Wheeler & Wheeler, 1930, the sole species included in 79
this genus. Here, the phylogeny of the Leptanillinae is inferred jointly from 10 protein-coding 80
genes, 28S rRNA, and 41 discrete male morphological characters under a Bayesian statistical 81
framework. This is the first combined-evidence Bayesian analysis to include the Leptanillinae 82
and is novel among studies of ant phylogeny in its inclusion of exclusively male morphological 83
characters (Barden et al. [2017] used both worker and male morphology in their Bayesian total-84
evidence inference). Despite the absence of nucleotide sequences for P. javana a total-evidence 85
approach facilitates the inclusion of this terminal and its confident phylogenetic placement. 86
Based upon the results of this joint molecular and morphological phylogenetic analysis, a revised 87
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Phylogeny of the Male-Based Ant Genus Phaulomyrma
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male-based definition of Leptanilla is provided, and Phaulomyrma is synonymized with that 88
genus. 89
Materials and Methods 90
Taxon Sampling 91
Thirty-eight terminals were included in total (Table 1). Discrete morphological data were scored 92
for those 35 terminals for which male material was available. These observations were 93
completed in November 2019, meaning that these did not include the recently described male of 94
Opamyrma hungvuong Yamane, Bui & Eguchi, 2008 (Yamada et al. 2020). In addition to O. 95
hungvuong, Anomalomyrma boltoni Borowiec, Schulz, Alpert & Baňar, 2011 and Leptanilla 96
revelierii Emery, 1870 were represented in this study by workers alone. The latter was included 97
on account of its status as the type species of that genus: regardless of future systematic revision 98
to the Leptanillinae, the concept of the genus Leptanilla will not exclude this species. DNA 99
sequences for the outgroup M. heureka were obtained from a worker ant, as published in 100
Borowiec et al. (2019). 101
Representatives of all male-based genera were included in total-evidence analyses, except for 102
Scyphodon. These include both Yavnella argamani Kugler, 1986 and Yavnella cf. indica, along 103
with two undescribed Yavnella morphospecies from Bhutan and Thailand, respectively; P. 104
javana and Phaulomyrma MM01, the latter morphospecies provisionally assigned to 105
Phaulomyrma by Boudinot (2015) and Borowiec et al. (2019); and a morphospecies of Noonilla 106
close to the type species Noonilla copiosa Petersen, 1968, along with three additional Noonilla 107
morphospecies identified as such according to the definition given by Petersen (1968). 108
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Phylogeny of the Male-Based Ant Genus Phaulomyrma
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Material is deposited in the following repositories: the Bohart Museum of Entomology, 109
University of California, Davis, CA, USA (UCDC); the California Academy of Sciences, San 110
Francisco, CA, USA (CASC); the California State Collection of Arthropods, Sacramento, CA, 111
USA (CSCA); the Lund Museum of Zoology, Lund, Sweden (MZLU); and the Australian 112
National Insect Collection, Canberra, Australia (ANIC). 113
Molecular Dataset 114
Phylogenetic inference was based upon 11 nuclear loci: 28S ribosomal DNA (28S), abdominal-A 115
(abdA), arginine kinase (argK), antennapedia (Antp), elongation factor 1-alpha F2 copy 116
(EF1αF2), long wavelength rhodopsin (LW Rh), NaK ATPase (NaK), DNA pol-delta (POLD1), 117
topoisomerase I (Top1), ultrabithorax (Ubx), and wingless (Wg). I derived these “legacy loci” for 118
19 terminals from the alignment of Borowiec et al. (2019) (doi: 10.5281/zenodo.2549806) but 119
expanded to include autapomorphic indels and introns, and constituting 11,090 bp. For further 120
detail on the protocols for the extraction and amplification of these genetic data, refer to Ward et 121
al. (2010) and Ward & Fisher (2016). I added 18 terminals to this “legacy-locus” intron-inclusive 122
dataset by retrieving orthologous loci from phylogenomic data acquired with the ultra-conserved 123
element (UCE) probe set hym-v2 (Branstetter et al. 2017), as follows. 124
In the case of these 18 terminals, DNA was extracted non-destructively using a DNeasy Blood 125
and Tissue Kit (Qiagen Inc., Valencia, CA) according to manufacturer instructions. DNA was 126
quantified for each sample with a Qubit 2.0 fluorometer (Life Technologies Inc., Carlsbad, CA). 127
Phylogenomic data were obtained from these taxa using the hym-v2 probe set, with libraries 128
being prepared and target loci enriched using the protocol of Branstetter et al. (2017). 129
Enrichment success and size-adjusted DNA concentrations of pools were assessed using the 130
SYBR FAST qPCR kit (Kapa Biosystems, Wilmington, MA) and all pools were combined into 131
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Phylogeny of the Male-Based Ant Genus Phaulomyrma
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an equimolar final pool. The contents of this final pool were sequenced by an Illumina HiSeq 132
2500 at the University of Utah’s High Throughput Genomics Facility. The FASTQ output was 133
demultiplexed and cleansed of adapter contamination and low-quality reads using 134
illumiprocessor (Faircloth 2013) in the PHYLUCE package. Raw reads were then assembled 135
with trinity v. 2013-02-25 (Grabherr et al. 2011). 136
The possibility of genetic contamination across UCE samples was examined by inferring the 137
phylogeny from a concatenated UCE alignment, unpartitioned, using IQ-Tree v. 1.6.10 (Nguyen 138
et al. 2015) on the CIPRES Science Gateway (v. 3.3) (Miller et al. 2010) with the GTR+G model 139
of nucleotide substitution for 1,000 ultrafast bootstrap replicates (Hoang et al. 2018): topologies 140
and branch lengths were plausible given preliminary morphological hypotheses, indicating that 141
contamination had not occurred. 142
Legacy loci orthologous with those used by Borowiec et al. (2019) were then recovered from 143
genome-scale data as follows with PHYLUCE (Faircloth 2016). I derived single exon sequences 144
representing each locus for Leptanilla GR02 from the alignment ANT-exon-sequences-40-taxa-145
reduced.fasta published by Branstetter et al. (2017), given the comparative completeness of the 146
matrix for that species, and its phylogenetic position nested well within the Leptanillinae. These 147
sequences were then used analogously to probes. Species-specific contig assemblies were 148
aligned using phyluce_assembly_match_contigs_to_probes.py (min_coverage = 50, min_identity 149
= 85), a list of legacy loci shared across all taxa was generated using 150
phyluce_assembly_get_match_counts.py, and separate FASTA files for each locus were created 151
using these outputs. Sequences were aligned separately by locus using MAFFT (Katoh et al. 152
2009) implemented with the command phyluce_assembly_seqcap_align.py, and these sequences 153
were then trimmed with Gblocks (Castresana 2000) as implemented by the wrapper script 154
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Phylogeny of the Male-Based Ant Genus Phaulomyrma
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phyluce_assembly_get_gblocks_trimmed_alignment_from_untrimmed.py (settings: b1 = 0.5, b2 155
= 0.5, b3 = 12, b4 = 7). Alignment statistics for the output FASTA files were calculated with 156
phyluce_align_get_align_summary_data.py. Finally, a matrix that was 80% complete with 157
respect to locus coverage was generated using the script 158
phyluce_align_get_only_loci_with_min_taxa.py. This contained only 6 out of the 10 protein-159
coding loci that I attempted to recover using the exon-based bioinformatic protocol of Branstetter 160
et al. (2017). Legacy loci recovered from UCE assemblies often included non-coding sequences 161
adjacent to the regions included in Borowiec et al. (2019), which were trimmed manually in 162
AliView. In whichever cases those loci had been recovered, sequences for the taxa represented 163
only in the dataset of Borowiec et al. (2019) were then aligned with the recovered legacy loci 164
using the online MAFFT interface (Katoh et al. 2019) with default settings. In cases where 165
legacy loci were not successfully recovered or were incomplete relative to preexisting Sanger-166
derived sequences, these loci were derived from the dataset of Borowiec et al. (2019) or, in the 167
case of Leptanilla GR03, Ward & Sumnicht (2012). These data were concatenated with UCE-168
derived sequences across all FASTA files, inasmuch all sequences for each morphospecies were 169
derived from the same specimen; and all loci were concatenated to produce a final alignment, 170
which was 9,011 bp in length. Further summary statistics for this final alignment are provided in 171
Table 1. P. javana was the only terminal for which molecular data were not obtained: this 172
species is known only from two slide-mounted syntypes collected in 1907. 173
Alignment was unambiguous once all loci were brought into their respective reading frames. 174
GenBank accession numbers for all loci used in this study, and NCBI accession numbers for the 175
raw UCE reads from which some loci were derived, are provided in Table 2. 176
Morphological Dataset 177
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Phylogeny of the Male-Based Ant Genus Phaulomyrma
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Forty-one discrete binary morphological characters were coded for all 35 morphospecies known 178
from males. All these specimens were examined with a Leica MZ75 compound microscope or by 179
reference to images on AntWeb, except for the male of M. heureka, in the case of which 180
observations were derived from Boudinot (2015: Figs. 11-12). I imaged specimens when 181
necessary using a JVC KY-F75 digital camera and compiled color photographs from these with 182
the Syncroscopy AutoMontage Program. Scanning electron microscopy was undertaken using a 183
Hitachi TM4000 tabletop microscope. Morphological terminology follows the Hymenoptera 184
Anatomy Ontology (Yoder et al. 2010), with some exceptions being derived from Bolton (2003) 185
and Boudinot (2018). The character coding scheme was binary and non-additive (Pleijel 1995). 186
Missing data were scored as ‘?’. Autapomorphic characters were included. Numerical scores for 187
all morphological characters are presented in Supplemental Table 1. 188
Non-additive binary coding has been criticized for its susceptibility to redundancy (Strong & 189
Lipscomb 1999), stipulation of compound characters, and the inadvertent conflation of 190
morphological absences that are not hierarchically equivalent (Brazeau 2011). These problems 191
largely result from careless character delimitation. I compensated for these potential flaws by 192
defining and using only characters that do not logically depend upon other characters. 193
Definitions of morphological character states are provided in Supplemental Material 1. 194
Phylogenetic Analyses 195
Best-fitting partitions of the molecular data, and substitution models for these partitions, were 196
inferred synchronously with PartitionFinder2 v. 2.1.1 (Guindon et al. 2010; Lanfear et al. 2012, 197
2017) on the CIPRES Science Gateway (Miller et al. 2010), with subsets being asserted a priori 198
according to locus and codon position. Introns were included. I+G general time-reversible 199
mixture models were excluded from consideration due to undesirable behavior under a 200
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Phylogeny of the Male-Based Ant Genus Phaulomyrma
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probabilistic framework (Yang 1996). As an alternative ad hoc partitioning scheme for 201
molecular data, I respectively partitioned all exonic loci so that 1st-2nd codon positions were 202
placed in their own partition separate from the 3rd, and modeled nucleotide substitution in all 203
partitions under GTR+G. Using AMAS (Borowiec 2016), the full 9,011-bp alignment was split 204
according to both partition schemes. 205
The Mkv model (Lewis 2001) was used to model substitution of morphological character states, 206
albeit with stationary frequencies of character states treated as free parameters (Felsenstein 1981) 207
in order to accommodate asymmetry in character state frequencies. Variation in evolutionary rate 208
among characters was accommodated by drawing rates from a gamma-distributed prior 209
probability distribution (+G), approximated with 8 discrete categories k. 210
Phylogenetic analyses were performed in a Bayesian statistical framework using RevBayes v. 211
1.0.11 (Höhna et al. 2017) compiled on Ubuntu Linux v. 13.04. Each analysis consisted of four 212
independent Markov chain Monte Carlo (MCMC) chains, each run for 50,000 generations. Trees 213
were sampled every 10 generations, with the first 25% of the run being discarded as burn-in. 214
MCMCs with respect to all continuous parameters were considered converged if the effective 215
sample sizes as given in Tracer v. 1.7.1 (Rambaut et al. 2018) were >200, with sufficiency of 216
MCMC mixing across posterior probability landscapes being qualitatively assessed using traces 217
of the respective log-likelihoods of each parameter across the course of the analysis. Maximum a 218
posteriori trees were compiled from this sample of each run, with node support expressed as 219
Bayesian posterior probability (BPP). 220
Data Availability and Nomenclature 221
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All nucleotide and morphological data along with PF2 configuration files, RevBayes scripts, and 222
output of both phylogenetic analyses, are available at the Dryad Digital Repository 223
(doi:10.25338/B8GP7C). Sequence Raw Archives (SRAs) of raw UCE reads, and UCE 224
assemblies, are publicly available on NCBI (Table 2). 225
This article has been registered in Zoobank (www.zoobank.org). The LSID number is 226
5F3BECF6-3715-47B3-8D0F-DE7D66E1DA0A. 227
Results 228
Bayesian total-evidence inference of leptanilline phylogeny under the two partitioning schemes 229
resulted in similar topologies, with none of the differences affecting the composition or 230
interrelationship of major clades, and therefore not influencing the generic status of 231
Phaulomyrma. The phylogeny inferred under the ad hoc partitioning scheme is provided on 232
Dryad (doi: 10.25338/B8GP7C). All discussion from here on will refer to the phylogeny inferred 233
under the scheme derived with PartitionFinder2 (Fig. 1). Most nodes in this phylogeny were 234
supported with BPP≥0.95 (Fig. 1). Those that were not were scattered and shallow, meaning that 235
the interrelationships among all major leptanilline clades are well-resolved. Although the 236
sampling of the Leptanillinae was more extensive than that of Borowiec et al. (2019), our 237
inferences were largely congruent. 238
The clade corresponding to the tribe Anomalomyrmini (labeled as Protanilla sensu lato in Fig. 239
1) is recovered with maximal support (BPP = 1), with A. boltoni sister to all sampled Protanilla 240
save Protanilla TH03—thus rendering Protanilla paraphyletic—moderately supported (BPP = 241
0.9009). Borowiec et al. (2019) recovered A. boltoni as sister to Protanilla TH03 with weak 242
support irrespective of statistical framework, albeit with more extensive sampling within the 243
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Phylogeny of the Male-Based Ant Genus Phaulomyrma
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Anomalomyrmini. However, the internal topology of the Anomalomyrmini does not have any 244
bearing upon the status of Phaulomyrma relative to other male-based leptanilline genera, nor its 245
status relative to Leptanilla. 246
Noonilla, Yavnella argamani and Yavnella cf. indica, Leptanilla revelierii, and Phaulomyrma 247
javana were firmly recovered within a clade corresponding to the Leptanillini (BPP = 1). As in 248
Borowiec et al. (2019) the Leptanillini bifurcate robustly, with Y. argamani (and Yavnella cf. 249
indica, which was not included in Borowiec et al. [2019]) recovered in a clade otherwise without 250
described representatives, which is hereinafter designated Yavnella sensu lato (BPP = 1). 251
Although morphologically diverse (Fig. 2), the male morphospecies that comprise the sister-252
group to Yavnella s. l. are distinguished from Yavnella s. l. by 1) clypeus with a medial axis no 253
longer than the diameter of the torulus, where the epistomal sulcus is distinct; and 2) pronotum 254
and mesoscutum that are not extended posteriorly in profile view. Since L. revelierii is recovered 255
within this clade, it is hereinafter referred to as Leptanilla sensu lato (BPP = 0.9999). 256
Leptanilla s. l. bifurcates into two well-supported clades: one is broadly Eurasian and Australian 257
in its representation (with a single Afrotropical representative included in this study), including 258
L. revelierii and P. javana (BPP = 0.9971); the other is Indo-Malayan, and includes Noonilla 259
(BPP = 0.9998). Of the 11 terminals recovered in this latter subclade, only Leptanilla TH01 was 260
included in Borowiec et al. (2019). Noonilla (BPP = 0.9769) is sister to a clade of highly 261
distinctive male morphospecies, recovered with maximal support (BPP = 1), that are 262
immediately recognizable by bizarre metasomal processes (heretofore hypothesized to be 263
extensions of the gonocoxae sensu Boudinot [2018] [Boudinot 2015: Fig. 10D]) and a comb-like 264
row of robust bristles on the protibia (Fig. 3). These morphospecies remain undescribed, but I 265
provisionally refer to the clade as the “Bornean morphospecies-group”: while present sampling is 266
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Phylogeny of the Male-Based Ant Genus Phaulomyrma
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too sparse to judge whether this clade is precinctive to Borneo, available material exclusively 267
originates on that island. The rather disparate morphospecies Leptanilla TH01 and Leptanilla 268
zhg-th01 are recovered as a clade with maximum support (BPP = 1), and this clade is in turn 269
sister to Noonilla + Bornean morphospecies-group. Leptanilla zhg-th01 is unique among the 270
Leptanillinae in possessing a recurved mesoscutellar horn (Fig. 4B). Since the type species of 271
Leptanilla (L. revelierii) is included within the Eurasian-Australian clade, this clade is 272
hereinafter referred to as Leptanilla sensu stricto. These two circumscriptions of the name 273
Leptanilla are supported by male morphology (see Discussion). 274
The support values of internal nodes within Leptanilla s. str. are generally poor, with the 275
placement of Leptanilla ZA01 and Leptanilla zhg-bt01 differing according to the partitioning 276
scheme. In either case the topology of Leptanilla s. str. is likely subject to strong stochastic error 277
due to the inclusion of P. javana, for which molecular data are entirely absent. While the 278
phylogeny of Leptanilla s. str. cannot be resolved with the inferential approach and dataset used 279
herein, the monophyly of this clade is robustly supported (BPP = 0.9971). The recovery of P. 280
javana within Leptanilla s. str. is therefore probable based upon Bayesian total-evidence 281
inference (see Discussion for description of morphological characters supporting this 282
conclusion). 283
The syntype of P. javana and the taxon dubbed “Phaulomyrma MM01 by Boudinot (2015) and 284
Borowiec et al. (2019) were recovered distant from one another in the leptanilline phylogeny 285
(Fig. 1). Total-evidence phylogenetic inference recovered the latter terminal within Yavnella s. l., 286
indicating that it was incorrectly assigned to Phaulomyrma by these authorities; another 287
undescribed male morphospecies referred to as Phaulomyrma by Boudinot (2015: Fig. 4F) was 288
not sequenced in this study, but conforms morphologically to Yavnella s. l., and so likewise was 289
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Phylogeny of the Male-Based Ant Genus Phaulomyrma
14
incorrectly identified as Phaulomyrma. Conversely, P. javana is here robustly recovered within 290
Leptanilla s. l., and moreover within Leptanilla s. str. While the internal phylogeny of Leptanilla 291
s. str. remains uncertain due to P. javana lacking molecular data, the monophyly of this clade 292
inclusive of Phaulomyrma is strongly supported by current findings. 293
Discussion 294
Delimitation of Subclades in Leptanillinae using Male Morphology 295
Male morphological characters corroborate phylogeny at nodes of variable depth. The four male 296
representatives of the Anomalomyrmini included in the present study can easily be distinguished 297
from male Leptanillini by the presence of a pterostigma (although wing venation may be 298
inaccessible due to deciduous wings in some male Leptanillini [pers. obs.]) and the absence of an 299
ocellar tubercle. Griebenow (in review) will provide a formal description of female-associated 300
male Protanilla and a male-based definition of the leptanilline tribes. Yavnella s. l. is likewise 301
well-supported by Bayesian total-evidence inference, as is Leptanilla s. l., with the former clade 302
diagnosed almost entirely by symplesiomorphies: the only putative autapomorphy of Yavnella s. 303
l. is concavity of the propodeum in profile view (Fig. 5A), which was previously noted by 304
Kugler (1986) as being distinctive to Yavnella. 305
Leptanilla s. str. is identifiable relative to other subclades of Leptanilla s. l. based upon the 306
following combination of male morphological characters: absence of posterior mesoscutellar 307
prolongation (observed in Leptanilla zhg-th01 and Leptanilla TH01); propodeum convex and 308
without distinct dorsal face (Fig. 5C); gonopodites articulated (otherwise among the Leptanillini 309
articulated only in Leptanilla zhg-th01 and some Noonilla); gonocoxae fully separated ventrally 310
(this character state [Fig. 6A] elsewhere observed among sampled Leptanillini in all Yavnella s. 311
l. except for “Leptanilla” TH03; and Leptanilla zhg-th01); and penial sclerites dorsoventrally 312
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Phylogeny of the Male-Based Ant Genus Phaulomyrma
15
compressed along their entire length, entire, and lacking sculpture (Fig. 7A), this character state 313
elsewhere observed in Leptanillini only among Yavnella s. l. (excluding “Leptanilla” TH03) and 314
Leptanilla zhg-th01. 315
Unlike Scyphodon, Noonilla, and even the male-based species Leptanilla palauensis (Petersen, 316
1968: p. 593), the status of Phaulomyrma as a leptanillineand as an anthas never been 317
debated. Wheeler & Wheeler (1930) established the genus based upon the presence of wing 318
veins and “unusually large genitalia” (Wheeler & Wheeler 1930: p. 193). Their argument 319
regarding wing venation has no merit, given that the forewing venation of P. javana falls within 320
the range of variation observed in putative Leptanilla morphospecies (Petersen, 1968: pp. 594-321
595), with all leptanilline males examined by Boudinot (2015) exhibiting at least one compound 322
abscissa on the forewing. Petersen (1968: p. 597) even referred to Leptanilla and Phaulomyrma 323
as “nearly identical” (when comparing these taxa to L. palauensis), but refrained from 324
synonymizing Leptanilla and Phaulomyrma on account of the apparent uniqueness of the 325
genitalia of P. javana as illustrated by Wheeler & Wheeler (1930: Figs. 2A, C). In passing, 326
Taylor (1965: p. 365) also mentioned Phaulomyrma as being “possibly synonymous” with 327
Leptanilla. 328
Examination of a syntype of P. javana (below designated as lectotype) demonstrates that its 329
genitalia are consistent with other sampled male Leptanilla s. str. to the exclusion of males 330
within the Indo-Malayan sister-group of Leptanilla s. str. (Fig. 8). Although the preservation of 331
this P. javana syntype on a slide prevents us from directly confirming stylar articulation, the 332
sharply recurved styli are consistent with the syndrome seen in dried male leptanillines with 333
articulated gonopodites (Kugler 1986; Ward and Sumnicht 2012), indicating that the gonopodites 334
are articulated in P. javana. Contra Fig. 2C of Wheeler & Wheeler (1930), the volsellae of P. 335
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Phylogeny of the Male-Based Ant Genus Phaulomyrma
16
javana are not discernible in situ (Fig. 8D). If their condition is truly “plate-like” as described by 336
Wheeler & Wheeler (1930: p. 196), the volsellae of P. javana resemble those observed in 337
undescribed Sicilian male morphospecies attributed to Leptanilla (Scupola & Ballarin 2009). 338
Dissection of Anatolian Leptanilla GR03, and Spanish material that closely resembles sequenced 339
males of Leptanilla s. str., demonstrates that the volsellae are likewise lamellate in these 340
morphospecies, having much the same condition as in Leptanilla africana (Baroni Urbani 1977: 341
Fig. 37) (not included in this study). Therefore, given the phylogeny of P. javana and its 342
morphological conformity to Leptanilla s. str. there is no justification for maintaining the genus 343
Phaulomyrma. It is perhaps a nomenclatural irony that Wheeler & Wheeler (1930: p. 193) note 344
that the derivation of the genus name is from the Greek phaulus, which they translate as “trifling 345
or paltry”: the justification for establishing Phaulomyrma as a genus was trifling indeed. 346
Leptanilla javana (Wheeler & Wheeler, 1930) comb. nov. 347
Fig. 8A-D. 348
Phaulomyrma javana Wheeler & Wheeler 1930: Figs. 1, 2C. 349
Phaulomyrma javana – Petersen 1968: 293. Figs. 16A-C. 350
Lectotype 351
INDONESIA • ; Jawa Barat, “Buitenzorg” [Bogor]; Mar. 1907; F.A.G. Muir leg.; MCZ 31142. 352
Paralectotype 353
Same data as for lectotype. 354
A complete male-based diagnosis of Leptanilla relative to other Leptanillinae under both broad 355
and strict circumscriptions of Leptanilla is provided below, with putative synapomorphies 356
represented in italics for the two circumscriptions. Only genital characters could be scored for 357
Leptanilla ZA01. 358
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Phylogeny of the Male-Based Ant Genus Phaulomyrma
17
Genus Leptanilla Emery, 1870 359
Type species: Leptanilla revelierii Emery, 1870: 196. 360
= Leptomesites Kutter, 1948 (286). Synonymy by Baroni Urbani, 1977 (433). Holotype 361
deposited at MHNG (Muséum d’Histoire Naturelle, Geneva). 362
= Phaulomyrma Wheeler & Wheeler, 1930 (193); syn. nov. Lecto- and paralectotype deposited 363
at MCZC (Museum of Comparative Zoology, Cambridge, Massachusetts). 364
Male Diagnosis of Leptanilla s. l. relative to other Leptanillinae 365
1. Mandibles articulated to gena (Fig. 9B). 366
2. Medial axis of clypeus no longer than diameter of torulus, when epistomal sulcus is 367
distinct. 368
3. Antennomere 3 shorter than scape. 369
4. Ocelli present and set on tubercle (Fig. 10) (with exception of Leptanilla [Bornean 370
morphospecies-group] zhg-my05). 371
5. Pronotum and mesoscutum posteriorly extended (Fig. 11B-C). 372
6. Notauli absent. 373
7. Pterostigma absent. 374
8. Propodeum not concave in profile view. 375
Male Diagnosis of Leptanilla s. str. relative to other Leptanilla s. l. 376
9. Anteromedian ocellus and compound eye not intersecting line parallel to dorsoventral 377
axis of cranium. 378
10. Profemoral ventral cuticular hooks absent. 379
11. Ventromedian protibial comb-like row of setae absent. 380
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Phylogeny of the Male-Based Ant Genus Phaulomyrma
18
12. Infuscation at juncture of Rf and 2s-rs+Rs+4-6 absent. 381
13. Antero-admedian line absent (HAO: 0000128). 382
14. Mesoscutellum not posteriorly prolonged. 383
15. Propodeum convex in profile view, without distinct dorsal face. 384
16. Abdominal sternite IX without posterolateral filiform processes. 385
17. Abdominal tergite VIII broader than long. 386
18. Gonocoxae medially separated*. 387
19. Gonopodites articulated. 388
20. Volsella lamellate, entire distally, without denticles*. 389
21. Penial sclerites dorsoventrally compressed, dorsomedian carina absent, ventromedian 390
carina sometimes present. 391
22. Phallotreme situated at penial apex, without vestiture. 392
*These character states observed so far as is possible with available specimens. 393
Notes. 394
1. The mandibles are fused to the gena (Fig. 9B) in sampled Yavnella s. l. except for 395
Leptanilla TH04: this character state is seen elsewhere in male ants only among some 396
Simopelta spp. (Ponerinae) (Brendon Boudinot, pers. comm.). 397
2. The epistomal sulcus is often difficult to distinguish in Leptanilla s. l., but the 398
anteroposterior reduction of the clypeus can be inferred by the situation of the toruli at 399
the anterior-most margin of the head (cf. Boudinot 2015: p. 30). 400
3. Antennomere 3 is longer than the scape in all sampled Yavnella s. l. except for 401
Leptanilla TH05. 402
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Phylogeny of the Male-Based Ant Genus Phaulomyrma
19
4. Ocelli are entirely absent in “Leptanilla” TH03 and Yavnella zhg-bt01. The ocellar 403
tubercle is absent in the Anomalomyrmini and O. hungvuong; within Leptanilla s. l. it is 404
absent in Leptanilla zhg-my05, which is here inferred to be a secondary loss. 405
5. As noted by Petersen (1968: p. 87), N. copiosa contrasts with other described male 406
Leptanillinae by the lack of an “elongated, laterally compressed” mesosoma. Yavnella 407
was described by Kugler (1986) as sharing this condition, which Petersen (1968) adduced 408
as plesiomorphic for the Leptanillinae. While the relative modification of the 409
mesosoma—here approximated by the proportions of the pronotum and mesoscutum—410
forms a morphocline across the male Leptanillinae, this morphocline is discontinuous, 411
with a gap between the morphospace occupied by Leptanilla s. l. (Fig. 11B-C) and that 412
occupied by O. hungvuong, the Anomalomyrmini, and Yavnella s. l. (Fig. 11A). Future 413
sampling of male Leptanillinae may close this gap in morphospace, which would limit 414
the diagnostic utility of pronotal and mesonotal length. 415
6. The absence of notauli is a synapomorphy of the tribe Leptanillini. The notauli in 416
Protanilla TH01 and Protanilla zhg-vn01, in the tribe Anomalomyrmini, are 417
homoplastically absent. 418
7. The absence of the pterostigma (Figs. 12A, C) is a synapomorphy of the Leptanillini. 419
8. The convexity of the propodeum in profile view is plesiomorphic for the Leptanillinae. 420
Its concave condition in Yavnella (Kugler 1986) is apomorphic for that genus. 421
9. The anteromedian ocellus is not situated orthogonally to the compound eye in profile 422
view in Leptanilla s. str. Leptanilla TH01 and zhg-th01, the Bornean morphospecies-423
group, and all examined Noonilla except for Noonilla zhg-my04. The concomitant 424
prognathy of the male cranium is unique among male Leptanillinae to Leptanilla s. l., and 425
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Phylogeny of the Male-Based Ant Genus Phaulomyrma
20
as adduced by Petersen (1968), this condition appears apomorphic among the 426
Leptanillinae. 427
10. A profemoral ventral cuticular hook (Fig. 13B) is unique among the morphospecies 428
sampled herein to Leptanilla (“Bornean morphospecies-group”) zhg-my02 and -05. 429
11. The ventromedian comb-like row of setae on the protibia is an autapomorphy of the 430
Bornean morphospecies-group. 431
12. The infuscation observed in the Bornean morphospecies-group at the juncture of Rf and 432
2s-rs+Rs+4-6 (Fig. 12C) is not enclosed anteriorly by an abscissa and appears to be 433
homoplasious with the pterostigma observed in male Anomalomyrmini. Infuscation of 434
the forewing is otherwise absent in the Leptanillini. 435
13. The antero-admedian line is present among the Leptanillini only among some Yavnella s. 436
l. and in Noonilla zhg-my04. 437
14. The mesoscutellum is posteriorly prolonged in Leptanilla TH01 and Leptanilla zhg-th01 438
(Fig. 4B). The differences in mesoscutellar shape between these morphospecies (see 439
Supplemental Material 1) are such that the homology of posterior mesoscutellar 440
prolongation is uncertain. 441
15. The propodeum has a distinct planar to depressed dorsal face in the Bornean 442
morphospecies-group (Fig. 5B). This condition is an autapomorphy of that clade. 443
16. The posterior margin of abdominal sternite IX is variously emarginate to entire in male 444
Leptanillinae or with a posteromedian process (e.g. Protanilla zhg-vn01, “Leptanilla445
TH03), but posterolateral filiform processes of abdominal sternite IX are an 446
autapomorphy of the Bornean morphospecies-group. 447
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Phylogeny of the Male-Based Ant Genus Phaulomyrma
21
17. Abdominal tergite VIII is longer than broad only in Noonilla (Fig. 14A), Scyphodon and 448
a bizarre male morphospecies from Côte d’Ivoire (CASENT0102373) for which 449
molecular data are unavailable. 450
18. The gonocoxae exhibit partial (Fig. 6B) to full (Fig. 6C) medial fusion at least in ventral 451
view in “Leptanilla” TH03, Noonilla, and all sampled members of the Bornean 452
morphospecies-group. Within Leptanilla s. l., complete lack of medial gonocoxal fusion 453
(Fig. 6A) is a symplesiomorphy of Leptanilla s. str., Leptanilla TH01, and Leptanilla 454
zhg-th01. 455
19. Articulation of the gonopodites encompasses both cases in which conjunctival membrane 456
is visible between the gonocoxa and stylus, and those in which the stylus is recurved 457
relative to the gonocoxa without apparent conjunctival membrane. This character state is 458
a symplesiomorphy of Leptanilla s. str., and among Leptanilla s. l. included in this study 459
is observed in Noonilla zhg-my01-2,6 and Leptanilla zhg-th01. 460
20. The volsellae cannot be observed without dissection in many male Leptanillinae (e.g., 461
Noonilla), limiting my ability to assess their condition. However, Leptanilla s. str. 462
contrast with the Anomalomyrmini, Yavnella s. l., and the Bornean morphospecies-group 463
in that the volsellae (where visible) are dorsoventrally flattened, entire, and lacking 464
sculpture (Fig. 15). This is one of only two synapomorphies of Leptanilla s. str. relative 465
to other Leptanilla s. l. 466
21. Dorsoventral compression at the penial apex is also observed in Yavnella s. l. (except for 467
Leptanilla” TH03). In the Indo-Malayan sister clade of Leptanilla s. str. the penial 468
sclerites are lateromedially compressed to subcircular, at least basally. Leptanilla zhg-469
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Phylogeny of the Male-Based Ant Genus Phaulomyrma
22
th01 exhibits an intermediate condition, with the penial apex being lateromedially 470
compressed and this condition less pronounced towards the base. 471
22. Position of the phallotreme with distal margin adjoining the penial apex appears to be 472
ancestral for the Leptanillini. The phallotreme is shifted basally in Leptanilla zhg-my02 473
and –5 (Fig. 16B), Noonilla, and Scyphodon. The outline of the phallotreme is subcircular 474
in these morphotaxa. Setae surrounding the phallotreme are observed in Noonilla and 475
Scyphodon; this character state is likely a synapomorphy of these genera. 476
Goals of Future Research 477
Two described male-based species of Leptanilla are worth noting here as requiring further study 478
and acquisition of fresh material: L. palauensis, which was transferred with some reservation to 479
Leptanilla from Probolomyrmex (Formicidae: Proceratiinae) by Taylor (1965); and Leptanilla 480
astylina. Examination of the holotype of L. palauensis demonstrates that according to the 481
morphological hypotheses made herein this species can be confidently referred to Leptanilla s. l., 482
but beyond that its affinities are unclear. Based upon available illustrations (Petersen 1968: Fig. 483
1) L. astylina likewise can be placed in Leptanilla s. l., and closely resembles Leptanilla s. str., 484
excluding its genitalia, which to judge from Petersen (1968) are unlike those of any specimen 485
that was examined in this study, and exclude it from the definition of Leptanilla s. str. given 486
herein. 487
The case of Scyphodon must also be briefly addressed here. Examination of a specimen 488
attributable to this monotypic male-based genus shows that it can be placed in Leptanilla s. l. As 489
reported by Petersen (1968), the genitalia of Scyphodon conspicuously resemble those of 490
Noonilla: there is no reason to conclude that Scyphodon belongs within Leptanilla s. str., and I 491
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Phylogeny of the Male-Based Ant Genus Phaulomyrma
23
predict that Scyphodon is either sister to, or nested within, Noonilla. Future total-evidence 492
Bayesian phylogenetic inference will resolve the relation of Scyphodon to other Leptanilla s. l. 493
Future acquisition and examination of novel material may necessitate revision of the male 494
diagnosis of Leptanilla provided here, but this diagnosis is robust to all morphological 495
observations made with sequenced material. As Yavnella s. l., Noonilla and the Bornean 496
morphospecies-group are known only from males, and L. revelierii is known only from female 497
castes, no argument can yet be made regarding the ranking of the former clades relative to 498
Leptanilla. Yavnella is here ranked as a genus, but the description of Yavnella workers may 499
reveal a morphological basis for subjective arguments for the subsumption of Yavnella within 500
Leptanilla. The delimitation of genera within the Leptanillini therefore depends not only upon 501
phylogenetic resolution of the many lineages known only from male material, but upon the 502
morphology of corresponding workers. Future molecular sequencing will be needed to associate 503
workers and/or gynes to leptanilline lineages that are known only from males: such an effort has 504
successfully linked Protanilla lini (Anomalomyrmini) with previously unassociated males 505
(Griebenow, in review). 506
Conclusions 507
I have here demonstrated the utility of discrete morphological data within a total-evidence 508
framework that includes molecular data in inferring the phylogeny of an ant taxon known only 509
from male morphology. Using probabilistic models, the phylogenetic position of P. javana is 510
robustly inferred in conjunction with taxa for which only molecular data, or both these and male 511
morphological data, are available. In that phylogeny, P. javana and L. revelierii are confidently 512
recovered within a subclade easily diagnosed by male morphological characters; disregarding 513
future retrieval of worker material and/or novel male specimens, Phaulomyrma can be 514
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Phylogeny of the Male-Based Ant Genus Phaulomyrma
24
synonymized with Leptanilla despite continued uncertainty in the bounds of the latter genus. 515
Future work will employ this Bayesian total-evidence approach to infer the affinity of other, 516
more peculiar leptanilline taxa for which molecular data are unavailable. With a robust 517
phylogeny inferred for the Leptanillinae that is congruent with male morphology, the parallel 518
taxonomy that bedevils this little-understood group of ants can begin to be resolved. 519
Conflict of Interest 520
The author declares no conflict of interest. 521
Acknowledgments 522
First and foremost, I must thank Ziad Khouri for his generosity in providing indispensable 523
assistance and conceptual advice on the writing of scripts for the Bayesian phylogenetic analyses 524
upon which this project relied. I thank Jadranka Rota (MZLU), Debbie Jennings (ANIC), Kevin 525
Williams (CSCA), and Brian Fisher (CASC) for loans of material for examination and non-526
destructive DNA extraction; Stefan Cover was of great help in facilitating access to a syntype of 527
P. javana. I also thank my present and past lab-mates Jill Oberski and Matt Prebus for their 528
diligent and painstaking work enriching UCEs from the specimens that were used in this study 529
(sometimes with my help, sometimes not). In the realm of data collection, I am grateful to Eli 530
Sarnat, Steve Heydon and Lynn Kimsey for allowing me the usage of equipment for this study; 531
the aid of Michael Branstetter in providing advice on the retrieval of legacy loci from UCE 532
datasets; and my labmate Brendon Boudinot, who was an enduring source of informative 533
feedback on the coding of morphological observation into discrete character states. Lastly, I must 534
thank my adviser Phil Ward for his invaluable feedback on the construction and finer details of 535
this manuscript, along with past work in acquiring the Sanger-sequenced data that I utilized in 536
this study, and for tutoring me in the delimitation of protein-coding loci from flanking introns. 537
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
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Phylogeny of the Male-Based Ant Genus Phaulomyrma
25
This research was supported by the University of California, Davis and by NSF grant DEB-538
1932405 to P. S. Ward. 539
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Yang, Z. (1996). Among-site rate variation and its impact on phylogenetic analyses. Trends in 715
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Phylogeny of the Male-Based Ant Genus Phaulomyrma
31
Fig. 1. Bayesian total-evidence phylogeny of the Leptanillinae under partitioning scheme 719
inferred with PartitionFinder2. Phylogeny was rooted a posteriori on Martialis heureka. Nodes 720
with BPP≥0.95 marked in red. (A) Protanilla zhg-vn01 (CASENT0842613); (B) “Leptanilla 721
TH08 (CASENT022755; Shannon Hartman); (C) Leptanilla zhg-my02 (CASENT0106416). 722
723
724
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Phylogeny of the Male-Based Ant Genus Phaulomyrma
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Fig. 2. Selected diversity of male Leptanilla s. l. (A) Leptanilla TH01 (CASENT0119792; April 725
Nobile); (B) Leptanilla zhg-bt02 (CASENT084612; not sequenced in this study); (C) Noonilla 726
zhg-my04 (CASENT0842610); (D) Leptanilla (Bornean morphospecies-group) zhg-my05 727
(CASENT0842571). Scale bar A = 0.2 mm.; B = 1 mm.; C-D = 0.5 mm. 728
729
730
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Phylogeny of the Male-Based Ant Genus Phaulomyrma
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Fig. 3. Protibia of Leptanilla zhg-my04 (CASENT0842555). Scale bar = 0.2 mm. 731
732
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Phylogeny of the Male-Based Ant Genus Phaulomyrma
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Fig. 4. Presence (B: Leptanilla zhg-th01; CASENT0842619) versus absence (A: Yavnella zhg-733
th01; CASENT0842621) of the posterior prolongation of the mesoscutellum in male Leptanillini. 734
Scale bar = 0.3 mm. 735
736
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Phylogeny of the Male-Based Ant Genus Phaulomyrma
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Fig. 5. Conditions of the propodeum in the Leptanilllinae. (A) Concave (Yavnella zhg-bt01; 737
CASENT0106384); (B) convex with distinct dorsal face (Leptanilla zhg-my02; 738
CASENT0106456); (C) convex without distinct dorsal face (Protanilla lini [OKENT0011097]; 739
male described by Griebenow, in review). 740
741
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
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Phylogeny of the Male-Based Ant Genus Phaulomyrma
36
Fig. 6. Ventral view of male genitalia across the Leptanillini. (A) Leptanilla ZA01 742
(CASENT0106354), (B) Noonilla zhg-my02 (CASENT0842595); (C) Leptanilla zhg-my04 743
(CASENT0842553). Scale bar A = 0.1 mm.; B = 0.3 mm.; and C = 0.5 mm. 744
745
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Phylogeny of the Male-Based Ant Genus Phaulomyrma
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Fig. 7. Dorsoventral (A) (Yavnella zhg-th01; CASENT0842620) vs. lateromedial (B) (Leptanilla 746
zhg-my04; CASENT0842553) compression of the penial sclerites in posterodorsal view. 747
Dorsomedian carina marked with arrow. Scale bar = 0.3 mm. 748
749
750
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Phylogeny of the Male-Based Ant Genus Phaulomyrma
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Fig. 8. Syntype of Phaulomyrma javana examined in this study (MCZ:Ent:31142). (A) Full-face 751
view; (B) profile view of mesosoma; (C) forewing; (D) metasoma and genitalia. Scale bar = 0.2 752
mm. 753
754
755
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
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Phylogeny of the Male-Based Ant Genus Phaulomyrma
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Fig. 9. Mandible of (A) Yavnella cf. indica (CASENT0106377) and (B) Leptanilla zhg-my03 756
(CASENT0842618). Scale bar A=0.03 mm.; B=0.05 mm. 757
758
759
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Phylogeny of the Male-Based Ant Genus Phaulomyrma
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Fig. 10. Full-face view of “Leptanilla” TH02 (CASENT0119531; Michele Esposito), with 760
ocellar tubercle marked. Scale bar = 0.1 mm. 761
762
763
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Phylogeny of the Male-Based Ant Genus Phaulomyrma
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Fig. 11. Profile of pronotum (black) and mesoscutum (blue) in male Leptanillini. (A) Yavnella 764
zhg-bt01 (CASENT0106384); (B) Noonilla zhg-my04 (CASENT0842610); (C) Leptanilla 765
(Bornean morphospecies-group) zhg-my03 (CASENT0106416). 766
767
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
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Phylogeny of the Male-Based Ant Genus Phaulomyrma
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Fig. 12. Examples of male forewing venation across the Leptanillinae. (A) Yavnella zhg-bt01 768
(CASENT0106384); (B) Protanilla zhg-vn01 (CASENT0842613); (C) Leptanilla (Bornean 769
morphospecies-group) zhg-my05 (CASENT0842571). 770
771
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
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Phylogeny of the Male-Based Ant Genus Phaulomyrma
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Fig. 13. Foreleg of (A) Yavnella argamani (CASENT0235253) and (B) Leptanilla zhg-id01 772
(CASENT0842626; not sequenced in this study). Scale bar=0.3 mm. 773
774
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
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Phylogeny of the Male-Based Ant Genus Phaulomyrma
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Fig. 14. Posterior view of abdominal tergite VIII in male Leptanillini. (A) Yavnella zhg-th01 775
(CASENT0842620) and (B) Noonilla zhg-my02 (CASENT0842592). Scale bar = 0.3 mm. 776
777
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
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Phylogeny of the Male-Based Ant Genus Phaulomyrma
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Fig. 15. Gonopodite and volsella (vol) of Leptanilla africana, sketched after Baroni Urbani 778
(1977: Fig. 37) by M. K. Lippey. Top of image is distal to body. 779
780
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
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Phylogeny of the Male-Based Ant Genus Phaulomyrma
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Fig. 16. Dorsoposterior view of phallotreme in (A) Leptanilla zhg-my04 (CASENT0842553); 781
ventroposterior view of phallotreme in (B) Leptanilla zhg-my05 (CASENT0106432). Scale bar 782
A = 0.3 mm.; B = 0.4 mm. 783
784
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
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Phylogeny of the Male-Based Ant Genus Phaulomyrma
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Table 1. Summary statistics for full 9,011-bp DNA sequence alignment. Generic assignments in 785
quotation marks were inherited from Borowiec et al. (2019). “ – ” = absent base; “ ? ” = 786
unknown base. 787
Taxon
Caste/Sex
Identifier
Percent
missing
AT
content
-
?
Anomalomyrma boltoni
Worker
CASENT0217032
16.613
0.525
1497
0
Leptanilla GR01
Male
CASENT0106060
17.434
0.536
1571
0
Leptanilla GR02
Male
CASENT0106236
16.879
0.539
1521
0
Leptanilla GR03
Male
CASENT0106058
7.424
0.529
668
1
Leptanilla TH01
Male
CASENT0119792
17.701
0.523
1595
0
Leptanilla” TH02
Male
CASENT0119531
16.558
0.513
1492
0
Leptanilla TH03
Male
CASENT0129721
16.68
0.521
1503
0
Leptanilla TH04
Male
CASENT0129695
16.702
0.512
1505
0
Leptanilla” TH05
Male
CASENT0134656
16.746
0.517
1509
0
Leptanilla” TH06
Male
CASENT0179537
16.857
0.511
1519
0
Leptanilla” TH08
Male
CASENT0227775
16.902
0.515
1523
0
Leptanilla TH09
Male
CASENT0227556
16.757
0.543
1510
0
Leptanilla ZA01
Male
CASENT0106085
16.824
0.545
1516
0
Leptanilla zhg-au01
Male
CASENT0758873
89.568
0.531
1435
6636
Leptanilla zhg-au02
Male
CASENT0758864
62.701
0.536
685
4965
Leptanilla zhg-th01
Male
CASENT0842614
60.448
0.545
801
4646
Leptanilla revelierii
Worker
CASENT0006788
51.038
0.552
536
4063
Leptanilla zhg-bt01
Male
CASENT0842617
64.421
0.517
862
4943
Leptanilla zhg-my02
Male
CASENT0106451
51.027
0.538
535
4063
Leptanilla zhg-my03
Male
CASENT0842618
52.558
0.544
673
4063
Leptanilla zhg-my04
Male
CASENT0842553
52.869
0.537
701
4063
Leptanilla zhg-my05
Male
CASENT0842568
55.499
0.529
619
4382
Martialis heureka
Worker
CASENT0106181
23.915
0.465
2155
0
Noonilla zhg-my01
Male
CASENT0842585
89.258
0.506
1396
6647
Noonilla zhg-my02
Male
CASENT0842599
63.489
0.532
470
5251
Noonilla zhg-my04
Male
CASENT0842610
81.212
0.542
1202
6116
Noonilla zhg-my06
Male
CASENT0106373
51.171
0.543
548
4063
Opamyrma hungvuong
Worker
CASENT0178347
16.347
0.477
1473
0
Phaulomyrma” MM01
Male
CASENT0179537
16.957
0.514
1528
0
Phaulomyrma javana
Male
MCZ:Ent:31142
100
0
9011
0
Protanilla TH01
Male
CASENT0119776
16.258
0.529
1465
0
Protanilla TH02
Male
CASENT0128922
16.28
0.529
1467
0
Protanilla TH03
Male
CASENT0119791
16.89
0.497
1522
0
Protanilla zhg-vn01
Male
CASENT0842613
60.803
0.521
228
5251
Yavnella argamani
Male
CASENT0235253
16.724
0.52
1507
0
Yavnella cf. indica
Male
CASENT0106375
61.98
0.516
1203
4382
Yavnella zhg-bt01
Male
CASENT0842616
50.838
0.524
518
4063
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted August 31, 2020. . https://doi.org/10.1101/2020.08.28.272799doi: bioRxiv preprint
Phylogeny of the Male-Based Ant Genus Phaulomyrma
48
Taxon
Caste/Sex
Identifier
Percent
missing
AT
content
-
?
Yavnella zhg-th01
Male
CASENT0842615
50.882
0.523
522
4063
788
789
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted August 31, 2020. . https://doi.org/10.1101/2020.08.28.272799doi: bioRxiv preprint
Phylogeny of the Male-Based Ant Genus Phaulomyrma
49
Table 2. NCBI and SRA accession numbers for DNA sequences used in this study. 790
Taxon
CASENT #
28S
AbdA
EF2
LwRh
Wg
AP
ArgK
NaK
POLD1
Top1
Ubx
Anomalomyrma boltoni
CASENT0217032
KU671445
KU672069
KU671496
KU671547
KU671598
KU671848
KU671656
KU672002
KU671925
KU671719
KU671782
Leptanilla GR01
CASENT0106236
EF012999
JN967847
JN967830
JN967890
JN967854
MF625736
JN967880
MF626276
MF625821
JN967820
JN967809
Leptanilla GR02
CASENT0106060
JN967864
JN967848
JN967831
JN967891
JN967856
MF625737*
JN967883
MF626277*
MF625822*
JN967823
JN967812
Leptanilla GR03
CASENT0106058
JN967868
JN967851
JN967834
JN967894
JN967859
N/A
JN967885
MT603718
MT526730
JN967826
JN967815
Leptanilla TH01
CASENT0119792
KU671447
JN967845
JN967836
KU671549
JN967853
KU671856
KU671660
KU672010
KU671933
KU671723
KU671786
Leptanilla” TH02
CASENT0119531
MF626108
MF625677
MF625890
MF626217
MF625999
MF625738
MF626161
MF626278
MF625823
MF626052
MF625943
Leptanilla TH03
CASENT0129721
MF626109
MF625678
MF625891
MF626218
MF626000
MF625739
MF626162
MF626279
MF625824
MF626053
MF625944
Leptanilla TH04
CASENT0129695
MF626110
MF625679
MF625892
MF626219
MF626001
MF625740
MF626163
MF626280
MF625825
MF626054
MF625945
Leptanilla” TH05
CASENT0134656
MF626111
MF625680
MF625893
MF626220
MF626002
MF625741
MF626164
MF626281
MF625826
MF626055
MF625946
Leptanilla” TH06
CASENT0179537
MF626112
MF625681
MF625894
MF626221
MF626003
MF625742
MF626165
MF626282
MF625827
MF626056
MF625947
Leptanilla” TH08
CASENT0227775
MF626113
MF625682
MF625895
MF626222
MF626004
MF625743
MF626166
MF626283
MF625828
MF626057
MF625948
Leptanilla TH09
CASENT0227556
MF626114
MF625683
MF625896
MF626223
MF626005
MF625744
MF626167
MF626284
MF625829
MF626058
MF625949
Leptanilla ZA01
CASENT0106085
AY867452
AY867468
EF013432
AY867483
AY867421
MF625745
JN967878
MF626285
MF625830
JN967818
JN967807
Leptanilla zhg-au01
CASENT0758873
N/A
N/A
N/A
N/A
N/A
N/A
MT526685
XXXXXXX
XXXXXXX
XXXXXXX
XXXXXXX
Leptanilla zhg-au02
CASENT0758864
N/A
N/A
N/A
N/A
N/A
N/A
MT526686
XXXXXXX
XXXXXXX
XXXXXXX
XXXXXXX
Leptanilla zhg-th01
CASENT0842614
N/A
N/A
N/A
N/A
N/A
N/A
MT526687
XXXXXXX
MT526731
XXXXXXX
XXXXXXX
Leptanilla revelierii
CASENT0006788
N/A
N/A
N/A
N/A
N/A
N/A
N/A
XXXXXXX
XXXXXXX
XXXXXXX
XXXXXXX
Leptanilla zhg-bt01
CASENT0842617
N/A
N/A
N/A
N/A
N/A
N/A
MT526684
XXXXXXX
MT526729
XXXXXXX
XXXXXXX
Leptanilla zhg-my02
CASENT0106451
N/A
N/A
N/A
N/A
N/A
N/A
MT526688
XXXXXXX
MT526732
XXXXXXX
XXXXXXX
Leptanilla zhg-my03
CASENT0842618
N/A
N/A
N/A
N/A
N/A
N/A
XXXXXXX
XXXXXXX
MT526734
XXXXXXX
XXXXXXX
Leptanilla zhg-my04
CASENT0842553
N/A
N/A
N/A
N/A
N/A
N/A
XXXXXXX
XXXXXXX
MT526735
XXXXXXX
XXXXXXX
Leptanilla zhg-my05
CASENT0842568
N/A
N/A
N/A
N/A
N/A
N/A
MT526687
XXXXXXX
MT526733
XXXXXXX
XXXXXXX
Martialis heureka
CASENT0106181
KU671448
KU672072
KU671499
KU671550
KU671601
KU671858
KU671661
KU672012
KU671935
KU671724
KU671787
Noonilla zhg-my01
CASENT0842585
N/A
N/A
N/A
N/A
N/A
N/A
XXXXXXX
XXXXXXX
XXXXXXX
XXXXXXX
XXXXXXX
Noonilla zhg-my02
CASENT0842599
N/A
N/A
N/A
N/A
N/A
N/A
XXXXXXX
XXXXXXX
XXXXXXX
XXXXXXX
XXXXXXX
Noonilla zhg-my04
CASENT0842610
N/A
N/A
N/A
N/A
N/A
N/A
XXXXXXX
XXXXXXX
XXXXXXX
XXXXXXX
XXXXXXX
*These accession numbers are erroneously attributed to CASENT0106067 (Leptanilla GR02) on 791
GenBank. 792
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted August 31, 2020. . https://doi.org/10.1101/2020.08.28.272799doi: bioRxiv preprint
... The latter concept, that is, an early split of Leptanillinae, has been consistently confirmed in molecular phylogenetic studies (Brady & al. 2006, Moreau & al. 2006, Branstetter & al. 2017, Borowiec & al. 2019, although with some uncertainty concerning the placement of M. heureka (see Rabeling & al. 2008, Kück & al. 2011, Moreau & al. 2013, Borowiec & al. 2019. Altogether, the Leptanillinae and Martialinae comprise nine genera and 70 valid species (Bolton 2020), although generic limits will be subject to revision (Borowiec & al. 2019, Griebenow 2020a). ...
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