On Irish stickleback:Morphological diversification in a secondary contact zone

Question: How parallel is adaptive evolution when it occurs from different genetic backgrounds? Background: Divergent evolutionary lineages of several post-glacial fish species including the threespine stickleback are found together in Ireland. Goals: To investigate the morphological diversity of stickleback populations in Ireland and assess whether morphology evolved in parallel between evolutionary lineages. Methods: We sampled stickleback from lake, river, and coastal habitats across Ireland. Microsatellite and mitochondrial DNA data revealed evolutionary history. Geometric morphometrics and linear trait measurements characterized morphology. We used a multivariate approach to quantify parallel and non-parallel divergence within and between lineages. Results: Repeated evolution of similar morphologies in similar habitats occurred across Ireland, concordant with patterns observed elsewhere in the stickleback distribution. A strong pattern of habitat-specific morphology existed even among divergent lineages. Furthermore, a strong signal of shared morphological divergence occurred along a marine-freshwater axis. Evidently, deterministic natural selection played a more important role in driving freshwater adaptation than independent evolutionary history. © 2013 Mark Ravinet.

On Irish sticklebacks: morphological diversification in a secondary contact zone

Question: How parallel is adaptive evolution when it occurs from different genetic backgrounds?
Background: Divergent evolutionary lineages of several post-glacial fish species including the threespine stickleback are found together in Ireland.
Goals: To investigate the morphological diversity of stickleback populations in Ireland and assess whether morphology evolved in parallel between evolutionary lineages.
Methods: We sampled stickleback from lake, river, and coastal habitats across Ireland. Microsatellite and mitochondrial DNA data revealed evolutionary history. Geometric morphometrics and linear trait measurements characterized morphology. We used a multivariate approach to quantify parallel and non-parallel divergence within and between lineages.
Results: Repeated evolution of similar morphologies in similar habitats occurred across Ireland, concordant with patterns observed elsewhere in the stickleback distribution. A strong pattern of habitat-specific morphology existed even among divergent lineages. Furthermore, a strong signal of shared morphological divergence occurred along a marine–freshwater axis. Evidently, deterministic natural selection played a more important role in driving freshwater adaptation than independent evolutionary history.

Structure and dynamics of hybrid zones at different stages of speciation in the common vole (Microtus arvalis)

The genetic structure and dynamics of hybrid zones provide crucial information for understanding the processes and mechanisms of evolutionary divergence and speciation. In general, higher levels of evolutionary divergence between taxa are more likely to be associated with reproductive isolation and may result in suppressed or strongly restricted hybridization. In this study, we examined two secondary contact zones between three deep evolutionary lineages in the common vole (Microtus arvalis). Differences in divergence times between the lineages can shed light on different stages of reproductive isolation and thus provide information on the ongoing speciation process in M. arvalis. We examined more than 800 individuals for mitochondrial (mtDNA), Y-chromosome and autosomal markers and used assignment and cline analysis methods to characterize the extent and direction of gene flow in the contact zones. Introgression of both autosomal and mtDNA markers in a relatively broad area of admixture indicates selectively neutral hybridization between the least-divergent lineages (Central and Eastern) without evidence for partial reproductive isolation. In contrast, a very narrow area of hybridization, shifts in marker clines and the quasi-absence of Y-chromosome introgression support a moving hybrid zone and unidirectional selection against male hybrids between the lineages with older divergence (Central and Western). Data from a replicate transect further support non-neutral processes in this hybrid zone and also suggest a role for landscape history in the movement and shaping of geneflow profiles.

Phylogeography and demographic history of Lacerta lepida in the Iberian Peninsula

The Iberian Peninsula is recognized as an important refugial area for species survival and diversification during the climatic cycles of the Quaternary. Recent phylogeographic studies have revealed Iberia as a complex of multiple refugia. However, most of these studies have focused either on species with narrow distributions within the region or species groups that, although widely distributed, generally have a genetic structure that relates to pre-Quaternary cladogenetic events. In this study we undertake a detailed phylogeographic analysis of the lizard species, Lacerta lepida, whose distribution encompasses the entire Iberian Peninsula. We attempt to identify refugial areas, recolonization routes, zones of secondary contact and date demographic events within this species. Results support the existence of 6 evolutionary lineages (phylogroups) with a strong association between genetic variation and geography, suggesting a history of allopatric divergence in different refugia. Diversification within phylogroups is concordant with the onset of the Pleistocene climatic oscillations. The southern regions of several phylogroups show a high incidence of ancestral alleles in contrast with high incidence of recently derived alleles in northern regions. All phylogroups show signs of recent demographic and spatial expansions. We have further identified several zones of secondary contact, with divergent mitochondrial haplotypes occurring in narrow zones of sympatry. The concordant patterns of spatial and demographic expansions detected within phylogroups, together with the high incidence of ancestral haplotypes in southern regions of several phylogroups, suggests a pattern of contraction of populations into southern refugia during adverse climatic conditions from which subsequent northern expansions occurred. This study supports the emergent pattern of multiple refugia within Iberia but adds to it by identifying a pattern of refugia coincident with the southern distribution limits of individual evolutionary lineages. These areas are important in terms of long-term species persistence and therefore important areas for conservation.

Sex-specific clines support incipient speciation in a common European mammal

Hybrid zones provide excellent opportunities to study processes and mechanisms underlying reproductive isolation and speciation. Here we investigated sex-specific clines of molecular markers in hybrid zones of morphologically cryptic yet genetically highly-diverged evolutionary lineages of the European common vole (Microtus arvalis). We analyzed the position and width of four secondary contact zones along three independent transects in the region of the Alps using maternally (mitochondrial DNA) and paternally (Y-chromosome) inherited genetic markers. Given male-biased dispersal in the common vole, a selectively neutral secondary contact would show broader paternal marker clines than maternal ones. In a selective case, for example, involving a form of Haldane’s rule, Y-chromosomal clines would not be expected to be broader than maternal markers because they are transmitted by the heterogametic sex and thus gene flow would be restricted. Consistent with the selective case, paternal clines were significantly narrower or at most equal in width to maternal clines in all contact zones. In addition, analyses using maximum likelihood cline-fitting detected a shift of paternal relative to maternal clines in three of four contact zones. These patterns suggest that processes at the contact zones in the common vole are not selectively neutral, and that partial reproductive isolation is already established between these evolutionary lineages. We conclude that hybrid zone movement, sexual selection and/or genetic incompatibilities are likely associated with an unusual unidirectional manifestation of Haldane’s rule in this common European mammal.

Contact zone dynamics and the evolution of reproductive isolation in a North American treefrog, the spring peeper (Pseudacris crucifer)

Despite over seven decades of speciation research and 25 years of phylogeographic studies, a comprehensive understanding of mechanisms that generate biological species remains elusive. In temperate zones, the pervasiveness of range fragmentation and subsequent range expansions suggests that secondary contact between diverging lineages may be important in the evolution of species. Thus, such contact zones provide compelling opportunities to investigate evolutionary processes, particularly the roles of geographical isolation in initiating, and indirect selection against hybrids in completing (reinforcement), the evolution of reproductive isolation and speciation. The spring peeper (Pseudacris crucifer) has six well-supported mitochondrial lineages many of which are now in secondary contact. Here I investigate the evolutionary consequences of secondary contact of two such lineages (Eastern and Interior) in Southwestern Ontario using genetic, morphological, acoustical, experimental, and behavioural evidence to show accentuated divergence of the mate recognition system in sympatry. Mitochondrial and microsatellite data distinguish these two lineages but also show ongoing hybridization. Bayesian assignment tests and cline analysis imply asymmetrical introgression of Eastern lineage nuclear markers into Interior populations. Male calls are divergent between Eastern and Interior allopatric populations and show asymmetrical reproductive character displacement in sympatry. Female preference of pure lineage individuals is also exaggerated in sympatry, with hybrids showing intermediate traits and preference. I suggest that these patterns are most consistent with secondary reinforcement. I assessed levels of post-zygotic isolation between the Eastern and Interior lineages using a laboratory hybridization experiment. Hybrid tadpoles showed equal to or greater fitness than their pure lineage counterparts, but this may be countered through competition. More deformities and developmental anomalies in hybrid tadpoles further suggest post-zygotic isolation. Despite evidence for pre-mating isolation between the two lineages, isolation appears incomplete (i.e. hybridization is ongoing). I hypothesize that potentially less attractive hybrids may circumvent female choice by adopting satellite behaviour. Although mating tactics are related to body size, genetic status may play a role. I show that pure Eastern males almost always engage in calling, while hybrids adopt a satellite tactic. An absence of assortative mating, despite evidence of female preference, suggests successful satellite interception possibly facilitating introgression.

When vicars meet: A narrow contact zone between morphologically cryptic phylogeographic lineages of the rainforest skink, Carlia rubrigularis

Phylogeographic analyses of the fauna of the Australian wet tropics rainforest have provided strong evidence for long-term isolation of populations among allopatric refugia, yet typically there is no corresponding divergence in morphology. This system provides an opportunity to examine the consequences of geographic isolation, independent of morphological divergence, and thus to assess the broader significance of historical subdivisions revealed through mitochondrial DNA phylogeography. We have located and characterized a zone of secondary contact between two long isolated (mtDNA divergence > 15%) lineages of the skink Carlia rubrigularis using one mitochondrial and eight nuclear (two intron, six microsatellite) markers. This revealed a remarkably narrow (width < 3 km) hybrid zone with substantial linkage disequilibrium and strong deficits of heterozygotes at two of three nuclear loci with diagnostic alleles. Cline centers were coincident across loci. Using a novel form of likelihood analysis, we were unable to distinguish between sigmoidal and stepped cline shapes except at one nuclear locus for which the latter was inferred. Given estimated dispersal rates of 90-133 m x gen(-1/2) and assuming equilibrium, the observed cline widths suggest effective selection against heterozygotes of at least 22-49% and possibly as high as 70%. These observations reveal substantial postmating isolation, although the absence of consistent deviations from Hardy-Weinberg equilibrium at diagnostic loci suggests that there is little accompanying premating isolation. The tight geographic correspondence between transitions in mtDNA and those for nuclear genes and corresponding evidence for selection against hybrids indicates that these morphologically cryptic phylogroups could be considered as incipient species. Nonetheless, we caution against the use of mtDNA phylogeography as a sole criterion for defining species boundaries.

Avifauna do estado do Acre: composição, distribuição geográfica e conservação

O estado do Acre faz fronteiras internacionais com o Peru e a Bolívia e nacionais com os estados do Amazonas e Rondônia. O Acre está localizado nas terras baixas da Amazônia sul‐ocidental, próximo ao sopé dos Andes, dentro de uma região considerada megadiversa da Amazônia brasileira. Apesar disso, a região ainda é pouco conhecida e considerada prioritária para a realização de novos levantamentos biológicos. Com o intuito de contribuir para o conhecimento da avifauna do sudoeste amazônico, este estudo teve como principais objetivos responder as seguintes questões: (a) Quantas e quais são as espécies de aves do estado do Acre? (b) Como as espécies estão distribuídas dentro do estado do Acre? e (c) Qual o estado de conservação das espécies residentes no estado do Acre? A metodologia para responder a estas questões contemplou: (a) uma ampla revisão bibliográfica; (b) dois anos de levantamento em campo, incluindo registros e a coletas de espécimes testemunhos; (c) a confecção do mapa de distribuição de cada táxon (incluindo espécies e subespécies); (d) a distribuição dos táxons pelas três grandes regiões interfluviais do Estado (leste, central e oeste); (e) a identificação de zonas de contato e hibridização, baseada na distribuição dos táxons parapátridos dentro do Estado; (f) o cálculo da distribuição potencial dos táxons dentro do Acre, baseado na extrapolação da área ocupada por cada unidade ecológica (fitofisionomia) onde eles foram registrados; (g) o cálculo da meta de conservação de cada táxon residente no Estado e (h) uma análise de lacunas, baseada na sobreposição dos mapas de distribuição potencial de cada táxon com o das Áreas Protegidas do Estado. A análise de lacuna foi realizada tendo três diferentes cenários como referência: (a) primeiro cenário ‐ levou em consideração todas as Áreas Protegidas; (b) o segundo cenário – levou em consideração apenas as Unidades de Conservação ‐ UCs de Proteção Integral e (c) terceiro cenário – levou em consideração apenas as UCs de Proteção Integral + as de Uso Sustentável (exclusas as Terras Indígenas). A revisão bibliográfica e os levantamentos (históricos e de campo) tiveram início em agosto de 2005 e se estenderam até dezembro de 2007. Após o término da revisão bibliográfica e das expedições em campo, foram compilados 7.141 registros de aves para o todo o estado do Acre. Destes, 4. 623 são de espécimes coletados, dos quais, 2.295 (49,6%) são oriundos de coletas feitas durante a realização deste estudo. Confirmou‐se para o Acre a presença de 655 espécies biológicas, distribuídas em 73 Famílias e 23 Ordens. Como consequência direta deste estudo, cinco novas espécies foram acrescentadas à lista de aves brasileiras. Registrou‐se também, 59 espécies migratórias, das quais, 30 (50,8%) são migrantes neárticas, 11 (18,6%) foram consideradas como migrantes intratropicais e 18 (30,5%) como migrantes austrais. De todas as espécies registradas no Estado, 44 são endêmicas do centro de endemismo Inambari. Dos 556 táxons de aves florestais residentes no Acre, 72,8% (405) distribui‐se nas três sub‐regiões do Estado; 10,0% (56) foi registrado apenas na sub‐região oeste; 5,3% (30) apenas na sub‐região leste e 0,5% (03) apenas na sub‐região central. Ao menos seis pares de táxons irmãos apresentaram padrão de distribuição alopátrida e 15 conjuntos de táxons apresentaram distribuição parapátrida dentro do Estado. Foram identificadas duas zonas de contato secundário (leste/oeste) e duas possíveis zonas de hibridização (leste/oeste) dentro do Estado. As análises de lacunas mostraram que no primeiro cenário, 87,1% dos táxons atingiram 100% da meta de conservação; 12% ficaram em classes de conservação intermediárias, ou seja, em lacuna parcial de proteção e apenas 0,8% ficaram em lacuna total de proteção. No segundo cenário, apenas 0,6% dos táxons atingiram 100% da meta de conservação; 97,6% ficaram em lacuna parcial e 1,8% ficaram em lacuna total de proteção. No terceiro cenário, 73,5% dos táxons atingiram 100% da meta de conservação; 25,5% ficaram em lacuna parcial e apenas 0,8% ficaram em lacuna total de proteção. As principais conclusões obtidas a partir deste estudo foram: (a) que a riqueza avifaunística do estado do Acre é bastante expressiva, porém, o número de espécies detectadas deverá aumentar à medida que novos levantamentos forem realizados; (b) que os rios Purus e Juruá não são as barreiras físicas que determinam o padrão de distribuição da maioria das aves residentes no estado do Acre; (c) que a presença de zonas de contato secundário, não coincidentes com o curso dos dois principais rios do Estado, dá suporte a ideia de que fatores não ligados a uma barreira física devem estar atuando na manutenção do padrão de distribuição atual de alguns táxons de aves residentes no Acre; (d) que o número de espécies “desprotegidas” ou em “lacuna parcial de proteção” entre a avifauna do Acre é muito baixo quando todo o sistema de Áreas Protegidas é levado em consideração, porém este número aumenta com a exclusão das Unidades de Conservação de Uso Sustentável e das Terras Indígenas; (e) que as aves restritas às campinas e campinaranas do oeste do Acre são as únicas que se encontram em lacuna do sistema de áreas protegidas do Acre, indicando a necessidade de se criar uma ou mais Unidades de Conservação para proteger este habitat específico.

Variação geográfica, zonas de intergradação e especiação no complexo Icterus Cayanensis-Chrysocephalus (Aves : Icteridae)

O complexo Icterus cayanensis-chtysocephalus apresenta um intrincado padrão de variação em plumagem e tamanho corpóreo. São reconhecidos, tradicionalmente, para o grupo seis táxons: Icterus chtysocephalus, I. cayanensis cayanensís. I. cayanensis tibialis, I. cayanensis tibialis, I. cayanensis valenciobuenoi, I. cayanensis periporphyrus e I. cayanensis pyrrhopterus, que se substituem geograficamente ao longo de grande parte da América do Sul. Neste estudo foi feita a descrição dos padrões de variação geográfica. Foram diagnosticadas quatro espécies, à luz do conceito filogenético de espécie: Icterus cayanensis (Amazônia Meridional), Icterus chrysocephalus (Amazônia Setentrional), Icterus tibialis (Caatinga) e Icterus pyrrhopterus (Chaco); os táxons I. cayanensis valenciobuenoi e I. cayanensis periporphyrus foram sinonimizados. Entre as formas amazonicas (chrysocephalus e cayanensis) foi detectada a presença de uma zona híbrida mais extensa do que aquela reportada na literatura. No Brasil Central foi diagnosticada a maior zona de intergradação conhecida para aves, com aproximadamente 2.300 km de extensão, produto do intercruzamento entre Icterus tibialis e Icterus pyrrhopterus, formas distribuídas pela Caatinga e Chaco, respectivamente. Postula-se que as zonas de intergradação diagnosticadas neste estudo são produto do intercruzamento de populações previamente diferenciadas em isolamento geográfico.

Tracing reinforcement through asymmetrical partner preference in the European common vole Microtus arvalis

Background The mechanistic basis of speciation and in particular the contribution of behaviour to the completion of the speciation process is often contentious. Contact zones between related taxa provide a situation where selection against hybridization might reinforce separation by behavioural mechanisms, which could ultimately fully isolate the taxa. One of the most abundant European mammals, the common vole Microtus arvalis, forms multiple natural hybrid zones where rapidly diverging evolutionary lineages meet in secondary contact. Very narrow zones of hybridization spanning only a few kilometres and sex-specific gene flow patterns indicate reduced fitness of natural hybrids and incipient speciation between some of the evolutionary lineages. In this study, we examined the contribution of behavioural mechanisms to the speciation process in these rodents by fine-mapping allopatric and parapatric populations in the hybrid zone between the Western and Central lineages and experimental testing of the partner preferences of wild, pure-bred and hybrid female common voles. Results Genetic analysis based on microsatellite markers revealed the presence of multiple parapatric and largely non-admixed populations at distances of about 10 km at the edge of the area of natural hybridization between the Western and Central lineages. Wild females from Western parapatric populations and lab-born F1 hybrids preferred males from the Western lineage whereas wild females of Central parapatric origin showed no measurable preference. Furthermore, wild and lab-born females from allopatric populations of the Western or Central lineages showed no detectable preference for males from either lineage. Conclusions The detected partner preferences are consistent with asymmetrical reinforcement of pre-mating reproductive isolation mechanisms in the European common vole and with earlier results suggesting that hybridization is more detrimental to the Western lineage. As a consequence, these differences in behaviour might contribute to a further geographical stabilization of this moving hybrid zone. Such behavioural processes could also provide a mechanistic perspective for frequently-detected asymmetrical introgression patterns in the largely allopatrically diversifying Microtus genus and other rapidly speciating rodents.

Incipient species formation in salamanders of the Ensatina complex

The Ensatina eschscholtzii complex of plethodontid salamanders, a well-known “ring species,” is thought to illustrate stages in the speciation process. Early research, based on morphology and coloration, has been extended by the incorporation of studies of protein variation and mitochondrial DNA sequences. The new data show that the complex includes a number of geographically and genetically distinct components that are at or near the species level. The complex is old and apparently has undergone instances of range contraction, isolation, differentiation, and then expansion and secondary contact. While the hypothesis that speciation is retarded by gene flow around the ring is not supported by molecular data, the general biogeographical hypothesis is supported. There is evidence of a north to south range expansion along two axes, with secondary contact and completion of the ring in southern California. Current research targets regions once thought to show primary intergradation, but which molecular markers reveal to be zones of secondary contact. Here emphasis is on the subspecies E. e. xanthoptica, which is involved in four distinct secondary contacts in central California. There is evidence of renewed genetic interactions upon recontact, with greater genetic differentiation within xanthoptica than between it and some of the interacting populations. The complex presents a full array of intermediate conditions between well-marked species and geographically variable populations. Geographically differentiated segments represent a diversity of depths of time of isolation and admixture, reflecting the complicated geomorphological history of California. Ensatina illustrates the continuing difficulty in making taxonomic assignments in complexes studied during species formation.

Cryptic diversity and evolutionary relationships among Australian closed-forest Melomys (Rodentia: Muridae) and related Australo-Papuan mosaic-tailed rats

This thesis investigates patterns of evolution in a group of native Australo-Papuan rodents. Past climatic change and associated sea level fluctuations, and fragmentation of wet forests in eastern Australia has facilitated rapid radiation, diversification and speciation in this group. This study adds to our understanding of the evolution of Australia’s rainforest fauna and describes the evolutionary relationships of a new genus of Australian rodent.

Impacts of climatic factors on evolution of molecular diversity and the natural distribution of wild stocks of the giant freshwater prawn (Macrobrachium rosenbergii)

Natural distributions of most freshwater taxa are restricted geographically, a pattern that reflects dispersal limitation. Macrobrachium rosenbergii is unusual because it occurs naturally in rivers from near Pakistan in the west, across India and Bangladesh to the Malay Peninsula, and across the Sunda Shelf and Indonesian archipelago to western Java. Individuals cannot tolerate full marine conditions, so dispersal between river drainage basins must occur at limited geographical scales when ecological or climatic factors are favorable. We examined molecular diversity in wild populations of M. rosenbergii across its complete natural range to document patterns of diversity and to relate them to factors that have driven evolution of diversity in this species. We found 3 clades in the mitochondrial deoxyribonucleic acid (mtDNA) data set that corresponded geographically with eastern, central, and western sets of haplotypes that last shared a common ancestor 1 × 106 y ago. The eastern clade was closest to the common ancestor of all 3 clades and to the common ancestor with its congener, Macrobrachium spinipes, distributed east of Huxley's Line. Macrobrachium rosenbergii could have evolved in the western Indonesian archipelago and spread westward during the early to mid-Pleistocene to India and Sri Lanka. Additional groups identified in the nuclear DNA data set in the central and western clades probably indicate secondary contact via dispersal between regions and modern introductions that have mixed nuclear and mtDNA genes. Pleistocene sea-level fluctuations can explain dispersal across the Indonesian archipelago and parts of mainland southeastern Asia via changing river drainage connections in shallow seas on wide continental shelves. At the western end of the modern distribution where continental shelves are smaller, intermittent freshwater plumes from large rivers probably permitted larval dispersal across inshore areas of lowered salinity.

Pleistocene climate fluctuations influence phylogeographical patterns in Melomys cervinipes across the mesic forests of eastern Australia

Aim Our aim was to clarify the lineage-level relationships for Melomys cervinipes and its close relatives and investigate whether the patterns of divergence observed for these wet-forest-restricted mammals may be associated with recognized biogeographical barriers. Location Mesic closed forest along the east coast of Australia, from north Queensland to mid-eastern New South Wales. Methods To enable rigorous phylogenetic reconstruction, divergence-date estimation and phylogeographical inference, we analysed DNA sequence and microsatellite data from 307 specimens across the complete distribution of M. cervinipes (45 localities). Results Three divergent genetic lineages were found within M. cervinipes, corresponding to geographically delineated northern, central and southern clades. Additionally, a fourth lineage, comprising M. rubicola and M. capensis, was identified and was most closely related to the northern M. cervinipes lineage. Secondary contact of the northern and central lineages was identified at one locality to the north of the Burdekin Gap. Main conclusions Contemporary processes of repeated habitat fragmentation and contraction, local extinction events and subsequent re-expansion across both small and large areas, coupled with the historical influence of the Brisbane Valley Barrier, the St Lawrence Gap and the Burdekin Gap, have contributed to the present phylogeographical structure within M. cervinipes. Our study highlights the need to sample close to the periphery of putative biogeographical barriers or risk missing vital phylogeographical information that may significantly alter the interpretation of biogeographical hypotheses.

Genetic divergence and echolocation call frequency in cryptic species of Hipposideros larvatus s.l. (Chiroptera : Hipposideridae) from the Indo-Malayan region

The intermediate leaf-nosed bat (Hipposideros larvatus) is a medium-sized bat distributed throughout the Indo-Malay region. In north-east India, bats identified as H. larvatus captured at a single cave emitted echolocation calls with a bimodal distribution of peak frequencies, around either 85 kHz or 98 kHz. Individuals echolocating at 85 kHz had larger ears and longer forearms than those echolocating at 98 kHz, although no differences were detected in either wing morphology or diet, suggesting limited resource partitioning. A comparison of mitochondrial control region haplotypes of the two phonic types with individuals sampled from across the Indo-Malay range supports the hypothesis that, in India, two cryptic species are present. The Indian 98-kHz phonic bats formed a monophyletic clade with bats from all other regional populations sampled, to the exclusion of the Indian 85-kHz bats. In India, the two forms showed 12–13% sequence divergence and we propose that the name Hipposideros khasiana for bats of the 85-kHz phonic type. Bats of the 98-kHz phonic type formed a monophyletic group with bats from Myanmar, and corresponded to Hipposideros grandis, which is suggested to be a species distinct from Hipposideros larvatus. Differences in echolocation call frequency among populations did not reflect phylogenetic relationships, indicating that call frequency is a poor indicator of evolutionary history. Instead, divergence in call frequency probably occurs in allopatry, possibly augmented by character displacement on secondary contact to facilitate intraspecific communication.