Unrecognized Species Diversity and New Insights Into Colour Pattern Polymorphism Within the Widespread Malagasy Snake Mimophis (Serpentes: Lamprophiidae)

Systematics and Biodiversity

ISSN: 1477-2000 (Print) 1478-0933 (Online) Journal homepage: http://www.tandfonline.com/loi/tsab20

Unrecognized species diversity and new insights into colour pattern polymorphism within the widespread Malagasy snake Mimophis (Serpentes: Lamprophiidae)

Sara Ruane, Edward A. Myers, Kahmun Lo, Sara Yuen, Rachel S. Welt, Maya Juman, India Futterman, Ronald A. Nussbaum, Gregory Schneider, Frank T. Burbrink & Christopher J. Raxworthy

To cite this article: Sara Ruane, Edward A. Myers, Kahmun Lo, Sara Yuen, Rachel S. Welt, Maya Juman, India Futterman, Ronald A. Nussbaum, Gregory Schneider, Frank T. Burbrink & Christopher J. Raxworthy (2017): Unrecognized species diversity and new insights into colour pattern polymorphism within the widespread Malagasy snake Mimophis (Serpentes: Lamprophiidae), Systematics and Biodiversity, DOI: 10.1080/14772000.2017.1375046 To link to this article: http://dx.doi.org/10.1080/14772000.2017.1375046

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Download by: [American Museum of Natural History] Date: 23 October 2017, At: 07:44 Systematics and Biodiversity (2017), 1–16

Research Article Unrecognized species diversity and new insights into colour pattern polymorphism within the widespread Malagasy snake Mimophis (Serpentes: Lamprophiidae)

SARA RUANE 1,2, EDWARD A. MYERS2, KAHMUN LO3, SARA YUEN3, RACHEL S. WELT2, MAYA JUMAN3, INDIA FUTTERMAN3, RONALD A. NUSSBAUM4, GREGORY SCHNEIDER4, FRANK T. BURBRINK2 & CHRISTOPHER J. RAXWORTHY2 1Department of Biological Sciences, 206 Boyden Hall, Rutgers University, 195 University Ave, Newark, NJ 07102, USA 2Department of Herpetology, American Museum of Natural History, Central Park West and 79th St., NY, NY 10024, USA 3Science Research Mentoring Program, American Museum of Natural History, Central Park West and 79th St., NY, NY 10024, USA 4Division of Reptiles and Amphibians, Museum of Zoology, Research Museums Center, 3600 Varsity Drive, University of Michigan, Ann Arbor, MI 48108, USA

(Received 16 May 2017; accepted 29 August 2017; published online 10 October 2017)

Although many wide-ranging taxa occur in Madagascar, phylogeographic studies for most of these species are still lacking. This is especially the case for snakes, where of more than 100 endemic species, the population structure of only two species has so far been examined. Here, we examine genetic population structure of one of the most common snakes of Madagascar, Mimophis mahfalensis (Grandidier, 1867). This taxon is the only representative of Psammophiinae in Madagascar, where the majority of species in this subfamily is distributed throughout mainland Africa. Applying an integrative approach, where both morphological data and genetic results from coalescent species delimitation analyses are considered, we determine that Mimophis mahfalensis is composed of two distinct taxa: M. mahfalensis in the central montane and southern parts of Madagascar, and a second new species restricted to the north and north-west, which we describe here. We also examine the colour pattern polymorphism exhibited in Mimophis, which has been previously hypothesized as sexually dimorphic and/or geographically correlated. However, we ﬁnd all three colour morphs in both sexes, and both species of Mimophis.Ourwork highlights the importance of phylogeographic studies that examine wide-ranging taxa to detect cryptic species diversity, even amongst species that are common, or have been previously considered to be well known.

www.zoobank.org/lsid:zoobank.org:pub:9791DC0B-49E5-4571-884C-4AA85EAF2472 Key words: African snakes, Colubroid, Lamprophiidae, Madagascar, Malagasy biodiversity, Psammophiinae, species delimitation

ranging taxa crossing multiple habitats. Of the existing Downloaded by [American Museum of Natural History] at 07:44 23 October 2017 Introduction phylogeographic studies on squamates of Madagascar, Madagascar is known for having an abundance of biodi- most are on lizards (Boumans, Vieites, Glaw, & Vences, versity and endemism (Myers, Mittermeier, Mittermeier, 2007; Chan, Choi, Raselimanana, Rakotondravony, & da Fonseca, & Kent, 2000), especially for squamate rep- Yoder, 2012; Florio, Ingram, Rakatondravony, Louis, & tiles, with >90% of the »400 species endemic to the Raxworthy, 2012; Florio & Raxworthy, 2016; Ratsoavina island (Uetz & Hosek, 2015). While recent studies have et al., 2010; Vences et al., 2004) with only two studies shown that there are microendemic species restricted to examining snake phylogeography: the Malagasy boas very small ranges (Raxworthy et al., 2008; Ruane, Bur- (Orozco-Terwengel, Nagy, Vieites, Vences, & Louis, brink, Randriamahtantsoa, & Raxworthy, 2016; Town- 2008) and the pseudoxyrhophiine Madagascarophis send, Vieites, Glaw, & Vences, 2009), there are (Nagy, Glaw, Andreone, Wink, & Vences, 2007). surprisingly few phylogeographic studies for wide- The majority of Madagascar’s terrestrial snake fauna is represented by the morphologically and ecologically Correspondence to: Sara Ruane. E-mail: [email protected]

diverse lamprophiid subfamily Pseudoxyrhophiinae (81 species, 82% of Malagasy snakes), as well as sanziniine boids (four species) and several lineages of typhlopoids (»13 species total). Additionally, there is a second lamprophiid subfamily on Madagascar represented by a single taxon, the monotypic Mimophis mahfalensis (Grandidier, 1867). Several studies have shown that this species is most closely related to mainland African psammophiines (Bogert, 1940; de Haan, 2003; Kelly, Barker, Villet, Broadley, & Branch, 2008; Nagy, Joger, Wink, Glaw, & Vences, 2003; Vidal & Hedges, 2002; Vidal et al., 2008). Mimophis represents the most recent natural dispersal of snakes into Madagascar (<13 million years ago; Samonds et al., 2013). The combination of the recent arrival and possible potential competitive exclusion from the diverse pseudoxyrhophiines species may account for the lack of species diversity within Mimophis. Mimophis mahfalensis has one of the largest range sizes for snakes in Madagascar, being absent from only the humid eastern coast (Glaw & Vences, 2007; Raxworthy, 2003). Mimophis mahfalensis is primarily terrestrial and can be found at high densities (Schutt, 2008) across a wide variety of habitats (Durkin, Steer, & Belle, 2011; Glaw & Vences, 2007; Ramanamanjato, Mcintyre, & Nussbaum, 2002), especially in anthropogenically disturbed areas (D’Cruze, Green, Robinson, & Gardner, 2006; Gardner & Jasper, 2009; McLellan, 2013; Ramana- manjato & Lehtinen, 2006), and is frequently associated with grasslands from which most other Malagasy snakes are absent (Bond, Silander, Ranaivonasy, & Ratsirarson, 2008). Mimophis mahfalensis is opisthoglyphic and its bite is known to cause non-lethal reactions in humans (Rosa et al., 2014). The venom of M. mahfalensis is probably used to subdue lizard prey (Domergue, 1989). While this snake is typically coloured grey and brown, M. mahfalensis exhibits intriguing colour pattern polymorphisms. There are three readily identified dorsal patterns: (1) a mostly patternless, uniform grey or tan, with finer, dark spotting or flecking on the dorsum, (2) a back- ground of grey or tan with a darker zig-zag pattern down the mid-dorsum, and (3) a dark brown or black stripe Downloaded by [American Museum of Natural History] at 07:44 23 October 2017 down the mid-dorsum, followed by a series of four contrasting lighter and darker dorso-lateral stripes before reaching the ventral scales (Fig. 1). It has been suggested Fig. 1. The three colour-pattern polymorphisms found in live these colour differences are due to sexual dichromatism, Mimophis, patternless (1.1), zig-zag (1.2), and striped (1.3). Pho- with females having the patternless grey/tan colouration, tos (1.1, 1.2) of Mimophis mahfalensis from Beraketa, south- and males having the zig-zag pattern (Domergue, 1969; eastern Madagascar and (1.3) of Mimophis occultus sp. nov. from Ankarana, north-western Madagascar, by CJR. Glaw & Vences, 1994, 2007) but this has never been formally tested. Furthermore, whether there is a geographic that these colour patterns could correspond to unrecog- component to these colour patterns has not been studied nized species or lineages within Mimophis. in detail, although the stripe pattern is considered typical Although Mimophis mahfalensis is currently considered a for the central plateau area and the subspecies, M. mahfa- single species, a previous phylogenetic study, based on five lensis madagascariensis (Glaw & Vences, 1994; Gunther, samples, suggested that this taxon may be composed of more 1868). Because there has been no detailed phylogeo- than one undescribed taxon (Kelly et al., 2008). Here, we graphic study examining these snakes, it is also possible examine population structure and morphology for M. Mimophis Systematics 3

mahfalensis across its complete range, and test for the pres- the program Tracer v.1.5 (Rambaut & Drummond, 2009) ence of cryptic species and the association with environment. to assess effective sample sizes of parameters, stationar- We then examine colour pattern distributions to determine if ity, and burnin for the BEAST analysis. this colouration polymorphism is related to either sexual Using the phased dataset for Mimophis samples, we ran dichromatism or geographically distinct lineages. the population clustering program Structure v.2.3.4 (Pritchard, Stephens, & Donnelly, 2000) to determine whether M. mahfalensis is a single, contiguous species or Materials and methods if there are multiple, discrete populations, possibly repre- Genetic samples senting cryptic taxa. Structure groups individuals into K populations by maximizing Hardy–Weinberg Equilibrium We sampled 39 individuals of Mimophis mahfalensis from and linkage equilibrium. We generated 20 runs for each K across Madagascar (32 localities with 1¡3 individuals per value, ranging from 1 to 5 populations for K, using the locality) and the outgroup Lamprophis guttatus for genetic admixture model and correlated allele frequencies. Each data (sample details in Appendix 1, see online supplemen- of these replicates were run for 106 generations, with a tal material, which is available from the article’s Taylor & burnin of 5 £ 105 generations. We determined the optimal Francis Online page at https://doi.org/10.1080/ value of K through examination of the Ln P(D) plot of 14772000.2017.1375046). Tissue samples included liver or each K value, which is the estimate of the posterior proba- muscle tissue preserved in EtOH and we extracted DNA D Ò bility of the data for a K value and the K Evanno method following the Qiagen DNEasy tissue protocol. We to determine the K value that captures the highest level of sequenced four loci in the forward and reverse directions: genetic structure (Evanno, Regnaut, & Goudet, 2005) the nuclear protein coding genes CMOS (509 bp), RAG2 using the web interface CLUMPAK (Kopelman, Mayzel, (645 bp), and the nuclear intron Nav 5 (512 bp), and the Jakobsson, Rosenberg, & Mayrose, 2015). mitochondrial gene, COI (662 bp), using the same primers Neither BEAST nor Structure explicitly test for species and conditions for PCR and sequencing conditions reported limits, therefore we used the results from the BEAST tree in Ruane, Raxworthy, Lemmon, Lemmon, and Burbrink Ò and the best estimated number of populations of Mimophis (2015). Contigs were assembled in Geneious using the from the Structure analyses (see Results) to test for these De Novo Assembly option and sequences subsequently lineage/populations as independent species using the aligned using the Geneious Alignment; sequences were Bayesian delimitation program BBP v.3.1 (Yang & Ran- trimmed so that the majority of individuals were the same nala, 2010, 2014). This method uses multiple loci and the length for each locus. The substitution model for each multi-species coalescent (Rannala & Yang, 2003; Yang, locus was determined using the Bayesian information crite- 2002) to determine if user-speciﬁed putative species are rion in jModelTest (Darriba, Taboada, Doallo, & Posada, supported as distinct, giving results in the form of poste- 2012). We used the program PHASE v.2.1 (Stephens & rior probability distributions (Pp) for either retaining or Donnelly, 2003; Stephens, Smith, & Donnelly, 2001)to collapsing nodes between the putative species. We tested determine the allele phase of nuclear loci for heterozygous species delimitations using both the mtDNA and nucDNA individuals. PHASE was run using the default parameters datasets as well as the nucDNA dataset alone, all under for 100 iterations, with a thinning interval of 1, and burnin the following conditions. We used the reversible-jump of 100; this analysis was run three times on each locus to Markov chain Monte Carlo (rjMCMC) algorithm 0 and ensure consistency amongst runs. The outputs for the most adjusted the ﬁne-tuning parameters between 0.30 and 0.70 probable pair of resulting alleles for the heterozygous indi- for each parameter to allow mixing of the rjMCMC viduals were used in subsequent analyses. amongst the possible species-delimitation models. Each Downloaded by [American Museum of Natural History] at 07:44 23 October 2017 BPP analysis was run for 100,000 generations, sampled every two generations, and with a 20,000 generation Population structure and species delimitation burnin. We varied the parameterizations to account for We used the Bayesian inference program BEAST v.1.8.3 both large and small population sizes combined with both (Drummond & Rambaut, 2007) to infer an initial deep and shallow divergence times, following the parame- concatenated tree of all loci, a nucDNA concatenated tree, terizations of Ruane et al. (2016). Each parameterization and individual gene trees to examine genetic structure was run three times with different starting seeds to ensure amongst the Mimophis samples. This unphased dataset consistency amongst results. was partitioned by locus and each used the most appropri- ate substitution model as determined using the Bayesian information criterion in jModelTest. We constrained Isolation by distance or environment Lamprophis guttatus as the outgroup taxon, used a Yule- To test if geographic distance or variation in climate across process speciation prior, and ran the analysis for 10 million Madagascar is important for structuring genetic variation generations, sampling every 1000 generations. We used across populations, we used redundancy analyses (RDA), 4 S. Ruane et al.

which is a constrained ordination method (Legendre & For- determining sex), we examined differences in morphology tin, 2010) performed in the statistical software program R between resulting Mimophis lineages. First, using R v.3.4.1 (R Core Team, 2013), using the scripts included in (R Core Team, 2013), we tested if resulting populations Myers, Hickerson, and Burbrink (2017). Current climatic differed significantly for ventral and subcaudal counts variables interpolated at 2.5 arc-min resolution were using Welch’s t-test, which provides a better estimate for obtained from the WorldClim database, which are 19 aver- unequal sample sizes or variance than does a standard t-test age monthly climatic data for minimum, mean, and maxi- (Ruxton, 2006; Welch, 1947). Subcaudal scale numbers are mum temperature and precipitation between the period of often different between sexes in snakes, we therefore parti- 1960–1990 (Hijmans, Cameron, Parra, Jones, & Jarvis, tioned the populations by sex, to determine if sexes could 2005), and extracted from the collection localities of individ- be pooled within lineages. To examine relative tail length uals with genetic data. We exclude Isothermality (BIO3) and differences between populations, we first tested for allome- Temperature Annual Range (BIO7) because they are tric growth for both males and females for each lineage to derived from other variables already included within the determine that we are only comparing adults. We used the WorldClim dataset. A genetic distance matrix was generated R package smatr 3 (Warton, Duursma, Falster, & Taskinen, from all genetic sequence data for both the concatenated 2012), which specifically tests for allometry using power dataset (39 individuals total) and the COI dataset (37 indi- functions. We used the sma function to determine if mea- viduals total) using the dist.dna function of the R package sured samples show isometric growth between tail length ape (Paradis, Claude, & Strimmer, 2004) with the GG95 (TL) and snout-vent length (SVL) using the power function model of sequence evolution (Galtier & Gouy, 1995)and log ðÞTL D aðlog ðÞSVL b where a is a proportionality coef- used as a response variable, where geographic distance ficient and b is a scaling exponent, and if significantly dif- between sampled localities (32 unique localities across all ferent from 1.0, would suggest that allometric growth is individuals) and current climate were the explanatory varia- present in our samples; TL and SVL are log transformed bles. Geographic distance and climate may be confounded prior to the analysis (see Warton et al., 2012 for details). by one another, therefore analyses incorporating only one of We then used the lm function in R to run a multiple linear these variables were conditioned on the other (using the regression to determine the significant predictors of tail Condition option within the rda function of vegan;Oksanen length (TL) to specifically determine if resulting lineages et al., 2007). We ran three different analyses with these vari- (specified here as species for the analysis) differed in tail ables, where predictions of genetic structure were tested lengths. We compared models that included the variables using distance, climate, and a model that included both dis- SVL, sex, species, the interaction of sex and species, the tance and climate. RDA analyses return an r2 value and an interaction of SVL and species, the interaction of SVL and ANOVA can be used to assess significance of the predictor sex, and the interaction of SVL, sex, and species as predic- variables in explaining genetic variation. tors of the response variable TL. Both TL and SVL were log-transformed to normalize the residuals of the model and improve the fit. Automated model selection was conducted using the dredge function of the MuMIn package v. 1.15.6 Morphology and colour patterns (Barton, 2016). Models were ranked using the Akaike Infor- Georeferenced records for a total of 239 Mimophis were mation Criterion with correction for small sample sizes used from the herpetology collection at the American (AICc). We used the anova function in R to determine if Museum of Natural History (AMNH), the University of high-ranking models were significantly different from each Michigan, Museum of Zoology (UMMZ), and the Univer- other and from the complete model including all predictors sity of Antananarivo (UADBA). Of these specimens, nine and possible interactions. Downloaded by [American Museum of Natural History] at 07:44 23 October 2017 did not have morphological vouchers available for exami- To score colour patterns, we examined all available nation but had tissues included in the genetic analyses specimens or field photos for colour pattern (n D 232). above, and two of these were also coded for colour pattern We scored the three readily identified dorsal patterns based on field photos. (Fig. 1): (1) uniform, (2) zig-zag, and (3) striped. Snakes We took morphological data from 30 Mimophis speci- that were unclear with respect to pattern due to poor pres- mens that had corresponding genetic data and examined an ervation or sloughing/loss of skin while in preservation additional 51 specimens distributed across the entire range were excluded from scoring. Using a Chi-square test of of Mimophis for the following morphological characters independence in R we determined whether these colour (to the nearest mm where applicable): total length, snout- patterns were distributed equally within resulting Mimo- vent length (SVL), tail length, ventral scale count, and sub- phis lineages (using only snakes that could be confidently caudal scale count. We also determined the relative tail assigned a pattern and to a lineage). To determine if any length of individuals using the ratio of tail length and SVL. colour pattern was restricted to a single sex, we sexed a Using sexed adults (n D 57 adult individuals total which sub-sample of specimens assigned to each colour morph included complete morphological data, see below on (17 uniform, 37 striped, and 50 zig-zag individuals), based Mimophis Systematics 5

on dissection and examining gonads or the presence of between the clades. These elevated within-clade divergen- everted hemipenes. All specimen information is provided ces were seemingly due to individuals with unshared sub- in Appendix 1 (see supplemental material online). stitutions and the high amounts of substructure within the southern clade (Fig. 2). There may also be even greater (species-level) substructure within the southern lineage Results that additional sampling would make clearer. Structure results (see supplemental material online) Genetic structure and species delimitation indicated that the Ln P(D) of populations increased sub- Lamprophis guttatus and all of the Mimophis individuals stantially from K D 1toK D 2, with diminishing increases were successfully sequenced for at least two of the four combined with high variance at higher K values; similarly, loci, with most sequenced for all four loci (see Appendix amongst the assessment of population changes (ΔK), K D 1; GenBank, accession numbers MF795180-MF795324). 2 was the optimal value of K using the Evanno method. JModelTest indicated that the best-ﬁt substitution models An examination of the Structure outputs for each run and to our data were: CMOS D HKY; RAG2 D F81; Nav K value shows that individuals assigned to the K D 2 intron 5 D HKY; COI D HKYCGamma. Structure populations had the highest assignment proba- All trees generated in BEAST had high ESS values for bilities, with 38 individuals assigned at >95%, and one all parameters (>200) and after assessment of stationarity individual assigned at >70%; these assignments generally in Tracer, 25% of samples were discarded as burnin. The corresponded to the same northern and southern popula- BEAST concatenated tree (Fig. 2) indicated two main tions of Mimophis inferred by the BEAST tree. As the clades of Mimophis, one with a northern and north-west- value of K increased beyond two, individuals from the ern distribution (n D 15) and one with a central and south- southern population were assigned to additional popula- ern distribution (n D 24). We compared the four tions with no obvious geographic pattern to the assign- individual nuclear gene trees to the concatenated tree ments, while those assigned to the northern population (additional trees are included as supplementary materials) typically remained within the same population for any and found no gene tree was identical to another but there value of K. However, UMMZ 237402 is strongly sup- was overall concordance amongst trees. Those trees from ported as sister to the rest of the northern clade in the RAG2 and CMOS were largely congruent, with individu- concatenated BEAST tree (Fig. 2), and is also suggested als falling into the northern or southern clades as they did independently when examining morphological data in the concatenated tree, with only one (CMOS) or two (Appendix 1), but is part of the southern population in the (RAG2) individuals differing in their placements (but not Structure results. This individual was successfully the same individuals). The intron Nav 5 has less similarity sequenced for only two loci (COI, CMOS); only CMOS in structure to the concatenated tree, with individuals provided any information for the Structure analyses as from the northern and southern lineages mixed, but still this individual has a private COI haplotype not shared showed some of the same overall signal/structure, e.g., a with any other Mimophis sequenced here. As the inference clade composed only of southern individuals. The COI of the tree in BEAST makes use of the sequence data from gene tree found all individuals were in the same two all available loci, combined with the independent morpho- clades (i.e., either northern or southern) as in the logical evidence that suggests it is part of the northern concatenated tree. The concatenated nuclear tree results clade (Appendix 1, see supplemental material online), we mirror the concatenated mtDNACnucDNA tree, adding considered it part of the northern lineage here for species additional support that the mtDNA is not solely driving delimitation purposes. Based on the two population the phylogenetic results (but see discussion on UMMZ assignments from Structure but including UMMZ 237402 Downloaded by [American Museum of Natural History] at 07:44 23 October 2017 237402 below). Minimum and maximum uncorrected as part of the northern clade, BPP analyses for each run, pairwise genetic divergences between the northern and using all datasets and under all parameterizations, resulted southern clades were: COI D 4.1¡8.0%; CMOS D in two well-supported species of Mimophis (Pp D 100% 0.2¡0.6%; RAG2 D 0.0¡0.4%; NAV intron 5 D in all runs for all parameterizations). Results from the 0.0¡0.9%. Uncorrected genetic distances (pairwise iden- RDA analyses demonstrate that neither geographic dis- tities) were on average greater between the northern and tance (COI: adjusted r2 D¡0.02, P-value D 0.49; southern lineages than within them for all loci. For exam- concatenated: adjusted r2 D 0.02, P-value D 0.2), current ple, for COI the mean pairwise difference between the climate (COI: adjusted r2 D¡0.02, P-value D 0.53; northern and southern clades was 6.3%, and the mean concatenated: adjusted r2 D 0.07, P-value D 0.15), or a pairwise differences within the northern and southern combination of these two variables (COI: adjusted r2 D clades were 0.7% and 3.3%, respectively. The within ¡0.08, P-value D 0.76; concatenated: adjusted r2 D 0.07, clade diversity of the southern lineage did have pairwise P-value D 0.11) signiﬁcantly explains genetic variation differences that were higher within the clade rather than across the distribution of Mimophis. 6 S. Ruane et al. Downloaded by [American Museum of Natural History] at 07:44 23 October 2017

Fig. 2. Concatenated BEAST tree of all loci for 39 Mimophis samples showing two main clades of Mimophis found in Madagascar, one with a northern and north-western distribution (n D 15) and one with a central and southern distribution (n D 24), with Lamprophis guttatus as an outgroup; indicate posterior probability support 95%.

Morphological differences in Mimophis between the two (n D 6); (Appendix 1, see supplemental material online). These assignments were ﬁrst made from Using all available data (genetic, morphological and/or geographic) we were able to categorize a total 239 Mimo- the results of the genetic analyses, giving us an estimate of the distributions for each putative species. Using indi- phis records as being members of a northern lineage (n D viduals that had both genetic and morphological data, we 46), southern lineage (n D 187), or an intermediate subsequently identiﬁed morphological differences Mimophis Systematics 7

Table 1. Morphological characteristics and geographic limits of Mimophis species.

Mimophis occultus sp. nov. Mimophis mahfalensis

All specimens All specimens Character Holotype n D 43 n D 32

Maximum SVL 443 mm 620 mm 580 mm Maximum tail length 172 mm 219 mm 191 mm Ratio Tail / SVL 0.39 0.33¡0.43 0.20¡0.36 Midbody dorsal scale rows 17 17 17 Ventral scales 160 152¡170 144¡167 Subcaudal scales 92 79¡104 44¡88 Uniform colour morph n/a yes yes Striped colour morph yes yes yes Zig-zag colour morph n/a yes yes Dark posterior eye stripe width 2 width 2 width > 2 temporal scales or temporal scales temporal scales fused to dark dorsal head Latitude range 16 12¡19 S18¡26 S Elevation range 41 m 0¡700 m 0¡1680 m

(described below) to diagnose specimens into the same southern lineages) as predictors, with the best-scoring groups as the genetic data. We then used this morphologi- model including just SVL and species (rank 1). The inter- cal diagnosis to assign specimens lacking genetic data action between SVL and species is included in the second into the northern or southern groups (n D 75 individuals model (rank 2) and sex is also included in the third model total). (rank 3). The analysis of the three most likely linear mod- We found consistent differences in the eye stripe col- els shows that (1) log(TL) increases significantly with log ouration feature between these two groups (see Table 1 (SVL) in all models (P < 0.0001 in all models), (2) log and the new species description for details). We also (TL) is significantly larger in the northern group than in found a significant difference for ventral counts between the southern group in all models (P < 0.0001 in all mod- the northern and southern populations (Welch’s t-test, els), and (3) when included in the model, the interaction North vs. South ventrals, t D 2.63, df D 25.45, P D between SVL and species is not a significant predictor of 0.015), with northern individuals having more ventrals on tail length (P D 0.4573) and that sex is also not a signifi- average compared with southern individuals (mean D cant predictor of tail length (P D 0.6212). These results 161, range D 152–170 North; mean D 157, range D 144– indicate that there is no sexual dimorphism in relative tail 167 South). For subcaudal scale counts, we found that the length, but there is a significant difference between the northern and southern populations were significantly dif- northern and southern groups. Relative tail length is ferent in subcaudal number (Welch’s t-test, North vs. greater in the northern population compared with the South subcaudals, t D 7.5, df D 26.04, P < 0.001), with southern (tail length/total length ratio: mean D 0.37, the northern lineage having higher numbers of subcaudal range D 0.33–0.43 North; mean D 0.30, range D 0.20– scales (mean D 94, range D 79–104 North; mean D 76, 0.36, South). Downloaded by [American Museum of Natural History] at 07:44 23 October 2017 range D 44–88 South). We also tested if subcaudal counts For colour pattern on the body, we found that specimens might be sexually dimorphic, and found no differences from the putative ranges of both the northern and southern between males and females in each population (Welch’s populations included all three patterns (Fig. 3). We ana- t-test, North males vs. females subcaudals, t D -0.44, df D lysed these on specimens that could only be confidently 22.24, P D 0.666; South males vs. females subcaudals, assigned to the northern or southern Mimophis clade (n D t D¡0.79, df D 11.08, P D 0.444). 223). The three colour patterns were not distributed equally We found no significant allometric effect with respect within the northern and southern lineages (x2 test of inde- to tail length for either sex in the northern or southern lin- pendence D 35.9; P <0.001; df D 2); there were fewer eages (P D 0.085–0.328 for all sexes from all lineages; zig-zag patterned snakes within the range of the northern outputs included as supplementary material), confirming population compared with the southern population, with that we are comparing only adult samples. For the multi- none observed in the extreme north of Madagascar ple regression analysis of tail length, the three most likely (Fig. 3). Striped snakes are absent in the southern and models (respective Akaike weights of 0.435, 0.176, south-western coastal regions, and the central High Plateau 0.149) all included SVL and species (the northern and lacked zig-zag patterned snakes (Fig. 3). Mimophis is likely 8 S. Ruane et al.

Fig. 3. (3.1) Sampling localities for genetic samples and morphological samples (total n D 89) and (3.2) colour pattern polymorphism localities for Mimophis specimens examined in this study that could be classiﬁed for colour pattern (n D 228) with examples of the three colour patterns from preserved Mimophis vouchers (locality details in Supplementary Appendix 1).

not sexually dichromatic; both sexes were found with all specimens collected by the Rev. W. Ellis, a missionary three pattern types (see Table 1 for a summary of morpho- who worked in the Central High Plateau of Madagascar. logical data, additional details are available in Appendix 1, Glaw & Vences (1994) recognized this subspecies based see supplemental material online). on the lined colouration pattern type given in the original description, which they thought was typical for Mimophis from Central Madagascar. Domergue (1969) also consid- Taxonomy of ered that these Central High Plateau Mimophis deserved Mimophis subspecific status based on their lined colouration, and Based on our genetic analyses and the corresponding mor- even assigned a name: M. mahfalensis lineatus. However, phological data, we find strong support for the presence of no type specimens were designated, and thus this taxon is two distinct species within Mimophis mahfalensis. The not considered valid (Brygoo, 1982). original description of Mimophis mahfalensis is based on Jourdran (1903) also reported a new variety of Mimo- specimens collected by Grandidier (1867) from the south- phis with an unusual white head that he referred to as west coast of Madagascar. Grandidier gives two localities: Mimophis albiceps in the text, and Mimophis mahfalensis ‘Machikova’, which likely corresponds to the present day albiceps in the associated illustration. However, no type Downloaded by [American Museum of Natural History] at 07:44 23 October 2017 Masikoro Port, now known as Androka at ¡25.029122 S, specimen or locality was designated with this description, 44.074371 E, and Anhoulabe, which is likely present day and this taxon has subsequently been ignored (Guibe, Ambohibe, a coastal town at ¡21.353016 S, 43.510772 E. 1958) or else considered a variety of Mimophis mahfalen- Both these localities are located within the range of the sis (Brygoo, 1982). We have never seen white-headed southern species found in our analyses. Thus this southern Mimophis in the field or collections, and strongly suspect species can be confidently assigned to Mimophis that this colouration feature is an artefact resulting from mahfalensis. fluid preservation. In alcohol, Mimophis specimens some- The southern species (Mimophis mahfalensis) also times shed epidermal skin on scales, revealing a much includes other previously described species and subspe- lighter colouration below. Thus, this white-headed speci- cies of Mimophis, collected in the central High Plateau men may have acquired this colouration simply by losing region of Madagascar. The putative subspecies M. mahfa- surface skin on its head, after it was preserved. lensis madagascariensis Gunther, 1868, which was given We therefore conclude that the northern species, as subspecific status from junior synonymy of M. mahfalen- identified by our results, represents an unnamed taxon, sis by Glaw & Vences (1994), was described from type which we formally describe below. Mimophis Systematics 9 Downloaded by [American Museum of Natural History] at 07:44 23 October 2017

Fig. 4. The holotype of Mimophis occultus sp. nov. UMMZ 237408. 10 S. Ruane et al.

Mimophis occultus sp. nov. Madagascar, Mahajanga Province (¡16.0212 S, 45.6029 E); UMMZ 222408 (Field Number RAX 0285) from Figs 1, 2, 3, 4; Table 1; Appendix 1(see supplemental Madagascar, Mahajanga Province (¡16.0176 S, 45.2718 material online) E); UMMZ 222407 (Field Number RAX 0215) from Madagascar, Mahajanga Province (¡16.4698 S, 45.3484 HOLOTYPE: UMMZ 237408 (RAN 68828 field num- E); UMMZ 237400 (Field Number RAN 67719) from ber), adult male, Madagascar, Mahajanga Province, Madagascar, Mahajanga Province (¡17.8944 S, 44.0168 Tsiambara Forest (¡15.920716 S, 45.9886 E), 41 m ele- E); UMMZ 222406 (Field Number RAX 0003) from vation, on 20 April 2002, collected by J. Spannring, J. Madagascar, Mahajanga Province (¡16.4698 S, 45.3484 Rafanomazantsoa, and M. Rakotoarivelo E); UMMZ 218446 (Field Number RAN 51981) from Madagascar, Mahajanga Province (¡14.8508 S, 48.2264 PARATYPES: UMMZ 209622 (RAN 38599 field num- E); AMNH pending (Field Number RAX 14073) from ber), juvenile male, Madagascar, Antsiranana Province, Madagascar, Mahajanga Province (¡15.4924 S, 46.6958 Montagne des Francais (¡12.3000 S, 49.3167 E), 150 m E); UMMZ 218447 (Field Number RAN 54286) from elevation, on 31 December 1991, collected by C. J. Rax- Madagascar, Mahajanga Province (¡18.7080 S, 44.7164 worthy, A. Raselimanana, and J.B. Ramanamanjato; E); UMMZ 219471 (Field Number RAN 55582) from UMMZ 209625 (RAN 38969 field number), juvenile Madagascar, Mahajanga Province (¡16.0430 S, 45.2597 female, Madagascar, Antsiranana Province, Ankarana E); UMMZ 237405 (Field Number RAN 68279) from Reserve, (¡12.9250 S, 49.1000 E), 119 m elevation, on 3 Madagascar, Mahajanga Province (¡16.0212 S, 45.6029 February 1992, collected by C.J. Raxworthy and A. Rase- E); UMMZ 237406 (Field Number RAN 68372) from limanana; AMNH 176427 (RAX 12588 field number), Madagascar, Mahajanga Province (¡15.9325 S, 45.7716 adult male, from Madagascar, Antsiranana Province, E); UMMZ 237401 (Field Number RAN 67844) from Ankarana Reserve (¡12.9311 S, 49.0558 E) on 7 Febru- Madagascar, Mahajanga Province (¡16.7655 S, 44.3805 ary 2014, collected by B. Randriamahtantsoa, C.J. Rax- E); UMMZ 237404 (Field Number RAN 68124) from worthy, and S. Ruane. Madagascar, Mahajanga Province (¡16.0205 S, 45.6036 E); UMMZ 237402 (Field Number RAN 68052) from NONTYPE MATERIAL EXAMINED: AMNH 140240 Madagascar, Mahajanga Province (¡16.1085 S, 45.0458 from Madagascar, Antsiranana Province (¡12.3736 S, E); UMMZ 237399 (Field Number RAN 67718) from 49.3669 E); UMMZ 209624 (Field Number RAN 38846) Madagascar, Mahajanga Province (¡17.8944 S, 44.0168 from Madagascar, Antsiranana Province (¡12.3000 S, E); UMMZ 240451 (Field Number RAN 69002) from 49.3167 E); UMMZ 209623 (Field Number RAN 38845) Madagascar, Mahajanga Province (¡13.0561 S, 48.9814 from Madagascar, Antsiranana Province (¡12.3000 S, E); UMMZ 240450 (Field Number RAN 68934) from 49.3167 E); AMNH 153327 (Field Number RAX 2250) Madagascar, Mahajanga Province (¡16.3584 S, 46.8259 from Madagascar, Antsiranana Province (¡13.7884 S, E); AMNH 160099 (Field Number RAX 9641) from 48.8 E); AMNH 153328 (Field Number RAX 4797) from Madagascar, Mahajanga Province (¡16.6298 S, 47.4161 Madagascar, Antsiranana Province (¡13.3727 S, 50.0029 E); UMMZ 218426 (Field Number RAN 49464) from E); UMMZ 240452 (Field Number RAN 70118) from Madagascar, Mahajanga Province (¡16.3136 S, 46.8173 Madagascar, Antsiranana Province (¡12.9707 S, 48.753 E); UMMZ 218425 (Field Number RAN 49449) from E); UMMZ 209679 (Field Number RAN 44306) from Madagascar, Mahajanga Province (¡16.3136 S, 46.8173 Madagascar, Antsiranana Province (¡13.7833 S, 48.3333 E); UMMZ 237391 (Field Number RAN 66985) from E); AMNH 176426 (Field Number RAX 12469) from ¡ ¡ Madagascar, Mahajanga Province ( 18.8725 S, 44.2399 Downloaded by [American Museum of Natural History] at 07:44 23 October 2017 Madagascar, Antsiranana Province ( 12.1907 S, 49.2197 E); UADBA pending (Field Number RAX 12471) from E); UMMZ 219470 (Field Number RAN 54964) from Madagascar, Antsirinana Province (¡12.1907 S, Madagascar, Antsiranana Province (¡12.8333 S, 49.5 E); 49.2697 E); UADBA pending (Field Number RAX UMMZ 219469 (Field Number RAN 54757) from Mada- 12408) from Madagascar, Antsirinana Province gascar, Antsiranana Province (¡12.8333 S, 49.5 E); (¡12.3342 S, 49.3581 E); UADBA pending (Field Num- UMMZ 218448 (Field Number RAN 54630) from Mada- ber RAX 12405) from Madagascar, Antsirinana Province gascar, Mahajanga Province (¡17.4908 S, 45.7464 E); (¡12.3342 S, 49.3581 E); UADBA pending (Field Num- UMMZ 218449 (Field Number RAN 54633) from Mada- ber RAX 11384) from Madagascar, Mahajanga Province gascar, Mahajanga Province (¡17.4360 S, 45.7531 E); (¡16.0750 S, 46.7312 E); see Appendix 1 (see supple- UMMZ 237398 (Field Number RAN 67632) from Mada- mental material online). gascar, Mahajanga Province (¡17.8944 S, 44.0168 E); UMMZ 237407 (Field Number RAN 68393) from Mada- ETYMOLOGY: The species epithet occultus is Latin for gascar, Mahajanga Province (¡15.9325 S, 45.7716 E); hidden or concealed, referring to the cryptic status of this UMMZ 237403 (Field Number RAN 68122) from taxon and having been hidden in plain sight. Mimophis Systematics 11

DIAGNOSIS: A species of Mimophis that can be diag- surface of the head is of a light tan/cream colour and nosed from M. mahfalensis by a combination of the fol- includes dark brown speckles or marks on the majority of lowing characteristics (see also Table 1): a narrow dark gular scales and the inframaxillaries. The dorsal pattern post-ocular eye stripe 2 temporal scales in width (vs. in consists of brown longitudinal stripes on a yellowish tan M. mahfalensis post-ocular eye stripe >2 temporal scales ground colour with a broad mid dorsal stripe and then 4 in width, or indistinct and fused with the dark dorsal head thinner stripes on each side of the body moving ventrally. colouration); 79¡104 subcaudal scales (vs. in M. mahfa- The dark brown central stripe starts anteriorly as two lensis 44¡88 subcaudal scales); and a generally larger tail blotches on the internasal scales of the head, separated by length/SVL ratio of 0.33¡0.43 (vs. in M. mahfalensis white on middle scales, and merging into one solid stripe 0.20¡0.36). approaching the neck and running down the dorsum on scale rows 8 (partially), 9, and 10; the stripe becomes bro- DESCRIPTION OF THE HOLOTYPE: Adult male in ken and less distinct approaching the posterior of the tail. good state of preservation, tail complete, removal of This central stripe includes a white centre that is only on dorso-lateral musculature from the posterior right side for the interior of the middle dorsal scale row (9), beginning DNA tissue sample, hemipenes not everted. Scales as a broken white line and becoming a solid line approxi- smooth, anal plate divided. Total length 615 mm, tail mately 1/6th of the way down the body, and becoming length 172 mm. Ventrals 160, subcaudals 92, divided. less distinct and fading entirely on the tail. On scale rows Head length (as jaw length) 17.7 mm, head width (widest 5 (partially), 6, 7 and 8 (partially) and counting from the point) 9.1 mm. Eyes large, 3.0 mm horizontal diameter, ventral scale up towards the dorsum mid body, the colour pupil round. On both left and right, supralabials 8, num- is yellowish tan, with occasional dark brown pigment bers 4 and 5 in contact with the eye. Infralabials 10, ﬁrst scattered on these scales. The single apical pit, encom- pair in contact behind mental, infralabials 1–5 in contact passed in a dot of dark brown pigment is especially promi- with inframaxillaries. Rostral broader than high, 2.6 mm nent on the scales in these rows. Scale rows 1, 2, and 3 wide/1.5 mm high, visible from above. Nasal entire in each has a very thin central dark stripe, lighter on the ante- contact with 1st and 2nd supralabials. Single loreal, in con- rior 1/3 of the body but becoming prominent on the poste- tact with nasal, internasal, prefrontal, and supralabial 2. rior 2/3 of the body. There is lighter brown pigment on Circumoculars 7, 1 supraocular, 2 preoculars, 2 subocu- the upper half of scale row 1 and the lower half of scale lars, and 2 postoculars; the preocular is divided so that a row 2 forming a faded light brown line. There is a thin lower triangular fragment is still in contact with the eye, stripe of light brown pigment on scale row 5, broken (or otherwise the circumocular count would be 6, with a sin- blotchy) anteriorly, but becoming prominent at mid body gle preocular. Temporals 2C3/1C3. Dorsal surface of where it drops to scale row 4 and forms a distinct stripe head includes a pair of internasals (width 1.5 mm/length posteriorly to the level of the anal scale, before breaking of suture 1.5 mm), pair of prefrontals (width 2.2/length of up again on the tail. The venter is cream coloured, espe- suture 1.9 mm), a pair of supraoculars (width cially anteriorly, but obscured posteriorly by profuse stip- 2.2 mm/length 4.1 mm), a frontal longer than wide (length pling of darker pigment, with pairs of thin dark brown 5.1 mm/anterior width 2.2 mm), and a pair of parietals marks on each ventral scale. (length of suture 3.6 mm). Two pairs of inframaxillaries separated by a mental groove (anterior inframaxillary VARIATION: Paratypes similar to the holotype except as length 4.0 mm/posterior inframaxillary length 4.2 mm), follows: UMMZ 209622, juvenile male, 477 mm total with the posterior pair separated posteriorly by a gular length, 135 mm tail length, ventrals 157, subcaudals 99, scale. Dorsal scale rows 17-17-13 at 10th ventral from number of preoculars 1, temporals 2C3 on both sides of Downloaded by [American Museum of Natural History] at 07:44 23 October 2017 anterior, midbody, and 10th ventral anterior to the cloaca. the head; UMMZ 209625, juvenile female, 551 mm total length, 166 mm tail length, ventrals 162, subcaudals 86 COLOURATION: The overall colouration faded in pres- (tail missing terminal tip), nasal divided below nostril, ervation, with the loss of the top layer on some scales. number of preoculars 1, temporals 2C3 on both sides of However the pattern remains clear and is of the ’striped’ the head (lower anterior temporal not in contact with post- variety. The dorsum of the head is a tannish ground colour ocular); AMNH 176427, adult male, 705 mm total length, with darker longitudinal markings on the internasals, pre- 180 mm tail length, ventrals 164, subcaudals 99, nasal frontals, supraoculars, frontals, and parietals. Dark brown divided below nostril, number of preoculars 1, temporals marks on the nasal, loreal, preocular and postocular scales 2C3 on both sides of the head, body colouration of the forms a dark stripe passing through the eye and fading patternless type. See Table 1 for a summary of variation posteriorly on the temporal scales. This dark post-ocular and comparison with M. mahfalensis. eye stripe is narrow, 2 temporal scales in width. Dark brown blotches are also present on all labial scales, with DISTRIBUTION AND NATURAL HISTORY: A species some blotches centred with pale brown spots. The ventral of Mimophis found from the northern tip of Madagascar, 12 S. Ruane et al.

throughout central and western Antsiranana Province, and Psammophiinae, included a northern Mimophis sample as far south as the towns of Maintirano, Ambatomainty, (from Montagne des Francais)¸ and four southerly samples and Tsaratanana in Mahajanga Province. The maximum (from Kirindy, Tulear, and Mount Ibity). Paralleling our known elevation recorded for this species is 700 m results the deepest genetic break for the genus was (UMMZ 218449, RAN 54633). There is a narrow zone between the single northern Mimophis and the four south- between Maintirano, Belo Tsiribihina, and Tsiroanoman- ern individuals. Kelly et al. (2008) concluded there was didy where several snakes have intermediate morphologi- likely unrecognized diversity in this genus requiring fur- cal characters (see Discussion), and where M. occultus ther investigation. While this prior study was not exten- and M. mahfalensis may co-occur (e.g., Cap Kimby). sive in its sampling of Mimophis, the similar results using Because we do not have genetic data for these morpholog- different individuals further support the findings of our ical intermediates, we cannot yet be sure of their taxo- study. nomic status. The presence of a cryptic taxon within the widespread Based on our field observations, we conclude that M. Mimophis may not be entirely unexpected, but surpris- occultus occurs in similar habitats and has similar behav- ingly, the three previously noted colour pattern polymor- iours as compared with M. mahfalensis. However, M. phisms are found in both species and are represented mahfalensis is distributed from sea level to much higher throughout much of the range of M. mahfalensis and M. elevations than M. occultus, reaching as high as 1680 m occultus (Fig. 3; Appendix 1). We find no support for the (Ibity). Mimophis occultus is common in disturbed areas putative High Plateau subspecies M. mahfalensis mada- (e.g., roadsides, grazed areas, and near human settle- gascariensis (see Glaw & Vences, 1994), which was pri- ments) and throughout open deciduous forested regions. It marily described based on having a striped colour pattern. is a diurnal and terrestrial snake, but we also observed Kelly et al. (2008) included the two putative subspecies in individuals at night at Montagne des Francais,¸ Ankaran- their sampling of Mimophis, and their resulting phylogeny dota, and Kelifely in low branches or on the ground; it is also did not show these subspecies formed monophyletic unclear if the snakes were active or preferred the low groups; the northern ’M. m. mahfalensis’ fell outside of branches as a roosting site. These snakes prey on squa- the clade including the southern ’M. m. mahfalensis’ sam- mates and probably frogs, as does M. mahfalensis.The ples. Rather than being a distinct lineage, the striped pat- dwarf chameleon Brookesia stumpfii was found in the tern is found within both of the Mimophis species, as are stomach of the paratype AMNH 176427. Several M. the patternless and zig-zag types (Fig. 3). These patterns occultus specimens (UMMZ 219470, 237400, 237403, are polymorphic within the genus and interestingly many 237404, 237405, 240451) were heavily infested with other taxa within Psammophiinae exhibit similar colour- worms. These appear to be sparagnosis infestations of ple- pattern polymorphisms including striped, unpatterned, rocercoid larvae, possibly of Spirometra tapeworms. Sim- and zig-zag forms, including: Psammophis crucifer, P. ilar infestations were also seen in M. mahfalensis notostictus, P. leightoni, P. sibilans, Psammophylax (e.g., UMMZ 209638); the source could be from infected rhombeatus (Branch, 1998); Hemirhagerrhis nototaenia, freshwater prey, such as frogs. Psammophylax variabilis (Spawls et al., 2001); Psammo- REMARKS: The large distribution and broad tolerance phis schokari (Kark, Warburg, & Werner, 1997). to human disturbance exhibited M. occultus makes it It has also been suggested that Mimophis is sexually unlikely to be of conservation concern. dimorphic with respect to these patterns, with males having the zig-zag and females the patternless colouration (Glaw & Vences, 2007). However, we find all three patterns are present in both sexes of Mimophis (Appendix 1). Downloaded by [American Museum of Natural History] at 07:44 23 October 2017 Discussion The potential adaptive significance or cause for these Considering the species diversity and large ranges of polymorphisms is unclear, but previous studies of snakes much of the snake fauna in Madagascar, it is surprising suggest that intraspecific colour pattern variation may be that few studies have investigated for potential cryptic related to anti-predator behaviours, ecologies, ontogenetic structure in Malagasy snakes. Using an integrated molecu- changes, general crypsis, body sizes, or thermoregulation, lar and morphological approach, we find that one of the either singly or in some combination (Forsman, 1995). most wide-ranging snakes in Madagascar, Mimophis mah- Although we can rule out ontogenetic changes and body falensis, is composed of two independently evolving line- size (these colour patterns are present in all size and onto- ages and describe the northern cryptic lineage as the new genetic classes), a detailed study isolating the other poten- species Mimophis occultus. tial causes is required to understand what is maintaining Our results are concordant with the only previously these polymorphisms within Mimophis. published Mimophis molecular phylogeny that included Mimophis occultus differs morphologically from M. five samples from across the island. Specifically, Kelly mahfalensis in two external features: (1) the dark post- et al. (2008), in estimating a general phylogeny for the ocular eye stripe, which is narrower on the temporal Mimophis Systematics 13

scales; and (2) the longer relative tail length (in both rivers will help determine if this river reduces gene flow sexes), which usually results in a higher number of sub- between the species. caudal scales and a larger tail length/SVL ratio. The eye It is likely that many other species in Madagascar will stripe feature is fully congruent with the molecular results. be revealed as complexes of distinct species; our research However, the possible adaptive function of eye stripes in highlights the need for phylogeographic examination of Mimophis (and indeed most snakes) has not yet been Malagasy snakes, especially those that are broadly distrib- investigated. The role of relative tail length in snakes has uted. Mimophis mahfalensis is perhaps the most common been previously studied (e.g., Guyer & Donnelly, 1990; diurnal snake encountered in most of Madagascar and our Lillywhite & Henderson, 1993), with shorter tails often analyses confirm that this widespread species is actually found in fossorial snakes and longer tails found in arboreal two distinct species, each with colour pattern polymor- snakes, and with primarily terrestrial species being some- phisms. We recommend further examination for Mimo- where in between (Feldman & Meiri, 2012). The relative phis and other snakes of Madagascar using phylogenomic tail length differences between the two species of Mimo- datasets, specifically with sampling across potential bar- phis is modest and likely reflects the similar terrestrial riers, as this will allow for better determination of which lifestyle of both taxa. geographic features are common drivers of diversification Interestingly, in the western region between Maintir- for Malagasy taxa. ano, Belo Tsiribihina, and Tsiroanomandidy where the two species may come in contact, we found six snakes with intermediate morphological characters. These snakes Acknowledgments have broad eye stripes on the temporal scales diagnostic Field studies in Madagascar were made possible due to the for M. mahfalensis, but also have the longer tails typical assistance of the Ministere de l’Environnement, de of M. occultus. Because we do not have tissues and molec- l’Ecologie et des Forêts, Madagascar National Parks, and ular data for these specimens, we are unable to determine the Universite d’Antananarivo, Departement de Biologie if these are unusually long-tailed M. mahfalensis,or Animale. Thanks to the American Museum of Natural whether they might represent hybrids. To make further History Ambrose Monell Cryo Collection (J. Feinstein) progress on resolving this issue will require making new and the Science Research Mentoring Program, M. Valen- collections in this region to acquire additional genetic cia Mestre for photos of the holotype, and S. Garnier for samples. Including genetic samples from this region in statistical discussion. future studies will allow for the examination of the degree of hybridization between the two taxa in contact zones. Speciation typically occurs via sympatry, parapatry, and allopatry, but while allopatry is the most frequently Disclosure Statement referenced mode of speciation, parapatry has been found The authors declare no conflicts of interest. to be more common that previously recognized (e.g., Fisher-Reid, Engstrom, Kuczynski, Stephens, & Wiens, 2013) Isolating geographic features, such mountain ranges Funding or rivers, as well as changes in ecotones/ecological gra- dients have been previously found important for specia- Funding provided by the National Science Foundation tion amongst Malagasy reptiles (Florio & Raxworthy, Division of Environmental Biology under [grant number 2016; Pearson & Raxworthy, 2009; Raxworthy, Ingram, DEB 1257610], [grant number 0641023], [grant number Rabibisoa, & Pearson, 2007). The potential barrier that 0423286], [grant number 9984496 (CJR)], [grant number Downloaded by [American Museum of Natural History] at 07:44 23 October 2017 separates the Mimophis sister taxa is uncertain, however 9625873], [grant number 9322600 (RAN, CJR)] and results from the RDA analyses rule out neutral divergence [grant number 1257926 (FTB)]; the American Museum of via geographic distance between populations and the Natural History Science Research Mentoring Program, potential effects of adaptation to current climates in driv- the Theodore Roosevelt Memorial Fund, the Richard ing speciation. There are several rivers that may contrib- Gilder Graduate School, and the Gerstner Scholar Pro- ute to divergence in the general region of the genetic gram. Research conducted at the American Museum of break and of morphological intermediacy for Mimophis Natural History and the Museum of Zoology, University mahfalensis and M. occultus. Specifically the Tsiribihina of Michigan. River, which has been found to impede gene flow in lemurs (Pastorini, Thalmann, & Martin, 2003) and has been suggested as a putative driver of speciation for sister taxa of Oplurus iguanas (Chan et al., 2012) may be an iso- Supplemental data lating geographic feature for these snakes. Targeted Supplemental information supporting this research can be found genetic sampling in this region for Mimophis across these online https://doi.org/10.1080/14772000.2017.1375046. 14 S. Ruane et al.

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