Gut microbial communities of hybridising pygmy angelfishes reflect species boundaries.

Hybridisation and introgression of eukaryotic genomes can generate new species or subsume existing ones, with direct and indirect consequences for biodiversity. An understudied component of these evolutionary forces is their potentially rapid effect on host gut microbiomes, and whether these pliable microcosms may serve as early biological indicators of speciation. We address this hypothesis in a field study of angelfishes (genus Centropyge), which have one of the highest prevalence of hybridisation within coral reef fish. In our study region of the Eastern Indian Ocean, the parent fish species and their hybrids cohabit and display no differences in their diet, behaviour, and reproduction, often interbreeding in mixed harems. Despite this ecological overlap, we show that microbiomes of the parent species are significantly different from each other in form and function based on total community composition, supporting the division of parents into distinct species, despite the confounding effects of introgression acting to homogenize parent species identity at other molecular markers. The microbiome of hybrid individuals, on the other hand, are not significantly different to each of the parents, instead harbouring an intermediate community composition. These findings suggest that shifts in gut microbiomes may be an early indicator of speciation in hybridising species.

Macroalgae exhibit diverse responses to human disturbances on coral reefs.

Scientists and managers rely on indicator taxa such as coral and macroalgal cover to evaluate the effects of human disturbance on coral reefs, often assuming a universally positive relationship between local human disturbance and macroalgae. Despite evidence that macroalgae respond to local stressors in diverse ways, there have been few efforts to evaluate relationships between specific macroalgae taxa and local human-driven disturbance. Using genus-level monitoring data from 1205 sites in the Indian and Pacific Oceans, we assess whether macroalgae percent cover correlates with local human disturbance while accounting for factors that could obscure or confound relationships. Assessing macroalgae at genus level revealed that no genera were positively correlated with all human disturbance metrics. Instead, we found relationships between the division or genera of algae and specific human disturbances that were not detectable when pooling taxa into a single functional category, which is common to many analyses. The convention to use percent cover of macroalgae as an indication of local human disturbance therefore likely obscures signatures of local anthropogenic threats to reefs. Our limited understanding of relationships between human disturbance, macroalgae taxa, and their responses to human disturbances impedes the ability to diagnose and respond appropriately to these threats.

The use of environmental DNA to monitor impacted coastal estuaries.

Environmental DNA (eDNA) metabarcoding is increasingly being used to assess community composition in coastal ecosystems. In this study, we chose to examine temporal and spatial changes in the aquatic community of Manly Lagoon - one of the most heavily developed and polluted estuaries in eastern Australia. Based on metabarcoding of the 16S mitochondrial gene (for fish) and the 18S nuclear gene (for macroinvertebrates), we identified seasonal differences in fish and macroinvertebrate community composition as well as species richness, which correlated, in some cases, with the environmental parameters of sea surface temperature and freshwater input. Moreover, given the greater taxonomic resolution of fish versus macroinvertebrate assignments, we identified several known migratory fish species of management importance that contributed significantly to the overall patterns observed. Overall, our data support the use of eDNA metabarcoding to track fish assemblages shifting in response to environmental drivers in polluted estuaries with increased sampling and consultation with historical data.

Regional versus latitudinal variation in the life-history traits and demographic rates of a reef fish, Centropyge bispinosa, in the Coral Sea and Great Barrier Reef Marine Parks, Australia.

Environmental temperature is an important determinant of physiological processes and life histories in ectotherms. Over latitudinal scales, variation in temperature has been linked to changes in life-history traits and demographic rates, with growth and mortality rates generally being greatest at low latitudes, and longevity and maximum length being greater at higher latitudes. Using the two-spined angelfish, Centropyge bispinosa, as our focal species, we compared growth patterns, growth rates, longevity, mortality, asymptotic length and maximum length across 22 reefs that span 13° of latitude within the Great Barrier Reef Marine Park (GBRMP) and the Coral Sea Marine Park (CSMP), Australia. We found no predictable latitudinal variation in mortality rates, growth patterns, growth rates, asymptotic or maximum length of C. bispinosa at regional to biogeographic scales. However, C. bispinosa consistently exhibited reduced longevity at lower, warmer latitudes within the CSMP. The greatest differences in mean maximum length of C. bispinosa were between continental (GBRMP) and oceanic (central CSMP) reefs of similar latitude, with individuals being larger on average on continental versus oceanic reefs. The lack of predictable life-history and demographic variation in C. bispinosa across a 13° latitudinal gradient within the CSMP, coupled with differences in mean maximum length between continental and oceanic reefs at similar latitudes, suggest that local environmental conditions have a greater influence than environmental temperature on the demographic rates and life-history traits of C. bispinosa.

Species integrity, introgression, and genetic variation across a coral reef fish hybrid zone.

Hybridization and introgression are evolutionarily significant phenomena breaking down species boundaries. "Hybrid zones" (regions of species overlap and hybridization) enable quantification of hybridization frequency and examination of mechanisms driving and maintaining gene flow. The hybrid anemonefish Amphiprion leucokranos is found where parent species (A. chrysopterus; A. sandaracinos) distributions overlap. Here, we examine geographic variation in hybridization and introgression, and potential impacts on parent species integrity through assessing relative abundance, social group composition, and genetic structure (mtDNA cytochrome b, 21 microsatellite loci) of taxa at three hybrid zone locations: Kimbe Bay (KB) and Kavieng (KA), Papua New Guinea; the Solomon Islands (SO). Relative abundances of and size disparities between parent species apparently drive hybridization frequency, introgression patterns, and genetic composition of taxa. Conspecific groups are most common in KB (65%) where parent species are similarly abundant. Conversely, mixed species groups dominate SO (82%), where A. chrysopterus is more abundant. Hybrids most commonly cohabit with A. sandaracinos in KB (17%), but with A. chrysopterus in KA (22%) and SO (50%). Genetic differentiation (nDNA) analyses indicate that parent species remain distinct, despite ongoing hybridization and hybrids are genetically similar to A. sandaracinos-resulting from persistent backcrossing with this smallest species. This study shows that hybridization outcomes may depend on the social and ecological context in which taxa hybridize, where relative abundance and disparate size of parent species explain the frequency and patterns of hybridization and introgression in the A. leucokranos hybrid zone, reflecting size-based dominance behaviors of anemonefish social groups.

Angels in disguise: sympatric hybridization in the marine angelfishes is widespread and occurs between deeply divergent lineages.

Hybridization events are not uncommon in marine environments where physical barriers are attenuated. Studies of coral reef taxa have suggested that hybridization predominantly occurs between parapatric species distributed along biogeographic suture zones. By contrast, little is known about the extent of sympatric hybridization on coral reefs, despite the large amount of biogeographic overlap shared by many coral reef species. Here, we investigate if the propensity for hybridization along suture zones represents a general phenomenon among coral reef fishes, by focusing on the marine angelfishes (family Pomacanthidae). Although hybridization has been reported for this family, it has not been thoroughly surveyed, with more recent hybridization studies focusing instead on closely related species from a population genetics perspective. We provide a comprehensive survey of hybridization among the Pomacanthidae, characterize the upper limits of genetic divergences between hybridizing species and investigate the occurrence of sympatric hybridization within this group. We report the occurrence of hybridization involving 42 species (48% of the family) from all but one genus of the Pomacanthidae. Our results indicate that the marine angelfishes are among the groups of coral reef fishes with the highest incidences of hybridization, not only between sympatric species, but also between deeply divergent lineages.

Population genomic response to geographic gradients by widespread and endemic fishes of the Arabian Peninsula.

Genetic structure within marine species may be driven by local adaptation to their environment, or alternatively by historical processes, such as geographic isolation. The gulfs and seas bordering the Arabian Peninsula offer an ideal setting to examine connectivity patterns in coral reef fishes with respect to environmental gradients and vicariance. The Red Sea is characterized by a unique marine fauna, historical periods of desiccation and isolation, as well as environmental gradients in salinity, temperature, and primary productivity that vary both by latitude and by season. The adjacent Arabian Sea is characterized by a sharper environmental gradient, ranging from extensive coral cover and warm temperatures in the southwest, to sparse coral cover, cooler temperatures, and seasonal upwelling in the northeast. Reef fish, however, are not confined to these seas, with some Red Sea fishes extending varying distances into the northern Arabian Sea, while their pelagic larvae are presumably capable of much greater dispersal. These species must therefore cope with a diversity of conditions that invoke the possibility of steep clines in natural selection. Here, we test for genetic structure in two widespread reef fish species (a butterflyfish and surgeonfish) and eight range-restricted butterflyfishes across the Red Sea and Arabian Sea using genome-wide single nucleotide polymorphisms. We performed multiple matrix regression with randomization analyses on genetic distances for all species, as well as reconstructed scenarios for population subdivision in the species with signatures of isolation. We found that (a) widespread species displayed more genetic subdivision than regional endemics and (b) this genetic structure was not correlated with contemporary environmental parameters but instead may reflect historical events. We propose that the endemic species may be adapted to a diversity of local conditions, but the widespread species are instead subject to ecological filtering where different combinations of genotypes persist under divergent ecological regimes.

Distinct patterns of hybridization across a suture zone in a coral reef fish (Dascyllus trimaculatus).

Hybrid zones are natural laboratories for investigating the dynamics of gene flow, reproductive isolation, and speciation. A predominant marine hybrid (or suture) zone encompasses Christmas Island (CHR) and Cocos (Keeling) Islands (CKE), where 15 different instances of interbreeding between closely related species from Indian and Pacific Oceans have been documented. Here, we report a case of hybridization between genetically differentiated Pacific and Indian Ocean lineages of the three-spot dascyllus, Dascyllus trimaculatus (Rüppell, 1829). Field observations indicate there are subtle color differences between Pacific and Indian Ocean lineages. Most importantly, population densities of color morphs and genetic analyses (mitochondrial DNA and SNPs obtained via RADSeq) suggest that the pattern of hybridization within the suture zone is not homogeneous. At CHR, both color morphs were present, mitochondrial haplotypes of both lineages were observed, and SNP analyses revealed both pure and hybrid genotypes. Meanwhile, in CKE, the Indian Ocean color morphs were prevalent, only Indian Ocean mitochondrial haplotypes were observed, and SNP analysis showed hybrid individuals with a large proportion (~80%) of their genotypes assigning to the Indian Ocean lineage. We conclude that CHR populations are currently receiving an influx of individuals from both ocean basins, with a greater influence from the Pacific Ocean. In contrast, geographically isolated CKE populations appear to be self-recruiting and with more influx of individuals from the Indian Ocean. Our research highlights how patterns of hybridization can be different at scales of hundreds of kilometers, due to geographic isolation and the history of interbreeding between lineages.

Social-environmental drivers inform strategic management of coral reefs in the Anthropocene.

Without drastic efforts to reduce carbon emissions and mitigate globalized stressors, tropical coral reefs are in jeopardy. Strategic conservation and management requires identification of the environmental and socioeconomic factors driving the persistence of scleractinian coral assemblages-the foundation species of coral reef ecosystems. Here, we compiled coral abundance data from 2,584 Indo-Pacific reefs to evaluate the influence of 21 climate, social and environmental drivers on the ecology of reef coral assemblages. Higher abundances of framework-building corals were typically associated with: weaker thermal disturbances and longer intervals for potential recovery; slower human population growth; reduced access by human settlements and markets; and less nearby agriculture. We therefore propose a framework of three management strategies (protect, recover or transform) by considering: (1) if reefs were above or below a proposed threshold of >10% cover of the coral taxa important for structural complexity and carbonate production; and (2) reef exposure to severe thermal stress during the 2014-2017 global coral bleaching event. Our findings can guide urgent management efforts for coral reefs, by identifying key threats across multiple scales and strategic policy priorities that might sustain a network of functioning reefs in the Indo-Pacific to avoid ecosystem collapse.

An examination of introgression and incomplete lineage sorting among three closely related species of chocolate-dipped damselfish (genus: Chromis).

AIM: To determine the impact of ecological and environmental histories on the evolution of coral reef damselfishes at two adjacent marine biogeographic suture zones. LOCATION: Indo-West Pacific, notably including two suture zones: Socotra and Christmas and Cocos/Keeling Islands. TAXON: Chromis dimidiata, Chromis margaritifer, and Chromis fieldi. METHODS: We utilized a combination of nuclear and mitochondrial genetic markers in addition to visual abundance survey data of these fishes. RESULTS: Despite genetic patterns consistent with incomplete lineage sorting and relatively low genetic differentiation among the three studied Chromis species, there is evidence of hybridization between C. margaritifer and C. fieldi at Christmas Island based on molecular and visual identification. Introgression appears to be spreading westwards to other C. fieldi populations based on COI haplotype comparison. Moreover, the genetic distance between C. margaritifer and C. fieldi suggests that Pleistocene sea-level fluctuations may have contributed to allopatric divergence and secondary contact between these two closely related species. MAIN CONCLUSIONS: Our study highlights that evolutionary processes in coral reef fishes operate differently between suture zones, possibly due to different ecological and environmental predispositions regulating secondary contact of sister species. While secondary contact likely led to hybridization and introgression at Christmas and Cocos/Keeling Islands, none of those processes seem present at Socotra for the chocolate-dipped damselfish. This difference is likely due to an environmental barrier caused by hydrodynamic regimes in the Gulf of Aden.

Ice ages and butterflyfishes: Phylogenomics elucidates the ecological and evolutionary history of reef fishes in an endemism hotspot.

For tropical marine species, hotspots of endemism occur in peripheral areas furthest from the center of diversity, but the evolutionary processes that lead to their origin remain elusive. We test several hypotheses related to the evolution of peripheral endemics by sequencing ultraconserved element (UCE) loci to produce a genome-scale phylogeny of 47 butterflyfish species (family Chaetodontidae) that includes all shallow water butterflyfish from the coastal waters of the Arabian Peninsula (i.e., Red Sea to Arabian Gulf) and their close relatives. Bayesian tree building methods produced a well-resolved phylogeny that elucidated the origins of butterflyfishes in this hotspots of endemism. We show that UCEs, often used to resolve deep evolutionary relationships, represent an important tool to assess the mechanisms underlying recently diverged taxa. Our analyses indicate that unique environmental conditions in the coastal waters of the Arabian Peninsula probably contributed to the formation of endemic butterflyfishes. Older endemic species are also associated with narrow versus broad depth ranges, suggesting that adaptation to deeper coral reefs in this region occurred only recently (<1.75 Ma). Even though deep reef environments were drastically reduced during the extreme low sea level stands of glacial ages, shallow reefs persisted, and as such there was no evidence supporting mass extirpation of fauna in this region.

Geography and island geomorphology shape fish assemblage structure on isolated coral reef systems.

We quantify the relative importance of multi-scale drivers of reef fish assemblage structure on isolated coral reefs at the intersection of the Indian and Indo-Pacific biogeographical provinces. Large (>30 cm), functionally-important and commonly targeted species of fish, were surveyed on the outer reef crest/front at 38 coral reef sites spread across three oceanic coral reef systems (i.e. Christmas Island, Cocos (Keeling) Islands and the Rowley Shoals), in the tropical Indian Ocean (c. 1.126 x 106 km2). The effects of coral cover, exposure, fishing pressure, lagoon size and geographical context, on observed patterns of fish assemblage structure were modelled using Multivariate Regression Trees. Reef fish assemblages were clearly separated in space with geographical location explaining ~53 % of the observed variation. Lagoon size, within each isolated reef system was an equally effective proxy for explaining fish assemblage structure. Among local-scale variables, 'distance from port', a proxy for the influence of fishing, explained 5.2% of total variation and separated the four most isolated reefs from Cocos (Keeling) Island, from reefs with closer boating access. Other factors were not significant. Major divisions in assemblage structure were driven by sister taxa that displayed little geographical overlap between reef systems and low abundances of several species on Christmas Island corresponding to small lagoon habitats. Exclusion of geographical context from the analysis resulted in local processes explaining 47.3% of the variation, highlighting the importance of controlling for spatial correlation to understand the drivers of fish assemblage structure. Our results suggest reef fish assemblage structure on remote coral reef systems in the tropical eastern Indian Ocean reflects a biogeographical legacy of isolation between Indian and Pacific fish faunas and geomorphological variation within the region, more than local fishing pressure or reef condition. Our findings re-emphasise the importance that historical processes play in structuring contemporary biotic communities.

Spatial and temporal patterns of mass bleaching of corals in the Anthropocene.

Tropical reef systems are transitioning to a new era in which the interval between recurrent bouts of coral bleaching is too short for a full recovery of mature assemblages. We analyzed bleaching records at 100 globally distributed reef locations from 1980 to 2016. The median return time between pairs of severe bleaching events has diminished steadily since 1980 and is now only 6 years. As global warming has progressed, tropical sea surface temperatures are warmer now during current La Niña conditions than they were during El Niño events three decades ago. Consequently, as we transition to the Anthropocene, coral bleaching is occurring more frequently in all El Niño-Southern Oscillation phases, increasing the likelihood of annual bleaching in the coming decades.

Biofluorescence as a survey tool for cryptic marine species.

As ecosystems come under increasing anthropogenic pressure, rare species face the highest risk of extinction. Paradoxically, data necessary to evaluate the conservation status of rare species are often lacking because of the challenges of detecting species with low abundance. One group of fishes subject to this undersampling bias are those with cryptic body patterns. Twenty-one percent of cryptic fish species assessed for their extinction risk (International Union for Conservation of Nature [IUCN]) are data deficient. We developed a nondestructive method for surveying cryptically patterned marine fishes based on the presence of biofluorescence (underwater biofluorescence census, UBC). Blue LED torches were used to investigate how widespread biofluorescence was in cryptic reef fishes in the Coral Triangle region. The effectiveness of UBC to generate abundance data was tested on a data-deficient pygmy seahorse species (Hippocampus bargibanti) and compared with data obtained from standard underwater visual census (UVC) surveys. We recorded 95 reef fish species displaying biofluorescence, 73 of which had not been previously described as biofluorescent. Of those fish with cryptic patterns, 87% were biofluorescent compared with 9% for noncryptic fishes. The probability of species displaying biofluorescence was 70.9 times greater for cryptic species than for noncryptic species. Almost twice the number of H. bargibanti was counted using the UBC compared with UVC. For 2 triplefin species (Ucla xenogrammus, Enneapterygius tutuilae), the abundance detected with UBC was triple that detected with UVC. The UBC method was effective at finding cryptic species that would otherwise be difficult to detect and thus will reduce interobserver variability inherent to UVC surveys. Biofluorescence is ubiquitous in cryptic fishes, making this method applicable across a wide range of species. Data collected using UBC could be used with multiple IUCN criteria to assess the extinction risk of cryptic species. Adopting this technique will enhance researchers' ability to survey cryptic species and facilitate management and conservation of cryptic marine species.

Genomic signatures of local adaptation reveal source-sink dynamics in a high gene flow fish species.

Understanding source-sink dynamics is important for conservation management, particularly when climatic events alter species' distributions. Following a 2011 'marine heatwave' in Western Australia, we observed high recruitment of the endemic fisheries target species Choerodon rubescens, towards the cooler (southern) end of its distribution. Here, we use a genome wide set of 14 559 single-nucleotide polymorphisms (SNPs) to identify the likely source population for this recruitment event. Most loci (76%) showed low genetic divergence across the species' range, indicating high levels of gene flow and confirming previous findings using neutral microsatellite markers. However, a small proportion of loci showed strong patterns of differentiation and exhibited patterns of population structure consistent with local adaptation. Clustering analyses based on these outlier loci indicated that recruits at the southern end of C. rubescens' range originated 400 km to the north, at the centre of the species' range, where average temperatures are up to 3 °C warmer. Survival of these recruits may be low because they carry alleles adapted to an environment different to the one they now reside in, but their survival is key to establishing locally adapted populations at and beyond the range edge as water temperatures increase with climate change.

Global warming and recurrent mass bleaching of corals.

During 2015-2016, record temperatures triggered a pan-tropical episode of coral bleaching, the third global-scale event since mass bleaching was first documented in the 1980s. Here we examine how and why the severity of recurrent major bleaching events has varied at multiple scales, using aerial and underwater surveys of Australian reefs combined with satellite-derived sea surface temperatures. The distinctive geographic footprints of recurrent bleaching on the Great Barrier Reef in 1998, 2002 and 2016 were determined by the spatial pattern of sea temperatures in each year. Water quality and fishing pressure had minimal effect on the unprecedented bleaching in 2016, suggesting that local protection of reefs affords little or no resistance to extreme heat. Similarly, past exposure to bleaching in 1998 and 2002 did not lessen the severity of bleaching in 2016. Consequently, immediate global action to curb future warming is essential to secure a future for coral reefs.

Naturally occurring hybrids of coral reef butterflyfishes have similar fitness compared to parental species.

Hybridisation can produce evolutionary novelty by increasing fitness and adaptive capacity. Heterosis, or hybrid vigour, has been documented in many plant and animal taxa, and is a notable consequence of hybridisation that has been exploited for decades in agriculture and aquaculture. On the contrary, loss of fitness in naturally occurring hybrid taxa has been observed in many cases. This can have negative consequences for the parental species involved (wasted reproductive effort), and has raised concerns for species conservation. This study evaluates the relative fitness of previously documented butterflyfish hybrids of the genus Chaetodon from the Indo-Pacific suture zone at Christmas Island. Histological examination confirmed the reproductive viability of Chaetodon hybrids. Examination of liver lipid content showed that hybrid body condition was not significantly different from parent species body condition. Lastly, size at age data revealed no difference in growth rates and asymptotic length between hybrids and parent species. Based on the traits measured in this study, naturally occurring hybrids of Chaetodon butterflyfishes have similar fitness to their parental species, and are unlikely to supplant parental species under current environmental conditions at the suture zone. However, given sufficient fitness and ongoing genetic exchange between the respective parental species, hybrids are likely to persist within the suture zone.

Angelfishes, Paper Tigers, and the Devilish Taxonomy of the Centropyge flavissima Complex.

The pygmy angelfishes (genus Centropyge) provide numerous examples of discordance between color morphology, taxonomy, and evolutionary genetic lineages. This discordance is especially evident in the Centropyge flavissima complex, which includes three primary color morphs, three previously recognized species (C. flavissima, Centropyge eibli, and Centropyge vrolikii) and three distinct mitochondrial (mtDNA) lineages that do not align with species designations. Our previous research showed that the putative C. flavissima arose independently in the Indian and Pacific Oceans, and the three mtDNA lineages align with geography rather than species assignments. Here, we add 157 specimens to the previous dataset of 291 specimens, spread across a greater geographic range, to pinpoint the distribution of mtDNA lineages and color morphs. We found that the mtDNA lineages show remarkably strong geographic boundaries corresponding to the Indian Ocean, Central-West Pacific, and Central-South Pacific. We also test the validity of the "Black Tiger Centropyge" in the C. flavissima species complex, a taxonomic oddity that is restricted to shoals and atolls off the coast of northwestern Australia, and the newly named Centropyge cocosensis assigned to the C. flavissima lineage in the Indian Ocean. We conclude that the Black Tiger Centropyge is not a valid species but rather an intermediate between sympatric color morphs that correspond to the putative species C. eibli and C. vrolikii Our greater sampling efforts also do not support the genetic distinctiveness of C. cocosensis given shared mtDNA haplotypes with the sympatric C. eibli and C. vrolikii, but instead we find conflicting lines of evidence concerning the taxonomy of this group. We urge caution and taxonomic restraint until the true nature of this species complex can be revealed.

Surgeons and suture zones: Hybridization among four surgeonfish species in the Indo-Pacific with variable evolutionary outcomes.

Closely related species can provide valuable insights into evolutionary processes through comparison of their ecology, geographic distribution and the history recorded in their genomes. In the Indo-Pacific, many reef fishes are divided into sister species that come into secondary contact at biogeographic borders, most prominently where Indian Ocean and Pacific Ocean faunas meet. It is unclear whether hybridization in this contact zone represents incomplete speciation, secondary contact, an evolutionary dead-end (for hybrids) or some combination of the above. To address these issues, we conducted comprehensive surveys of two widely-distributed surgeonfish species, Acanthurus leucosternon (N=141) and A. nigricans (N=412), with mtDNA cytochrome b sequences and ten microsatellite loci. These surgeonfishes are found primarily in the Indian and Pacific Oceans, respectively, but overlap at the Christmas and Cocos-Keeling Islands hybrid zone in the eastern Indian Ocean. We also sampled the two other Pacific members of this species complex, A. achilles (N=54) and A. japonicus (N=49), which are known to hybridize with A. nigricans where their ranges overlap. Our results indicate separation between the four species that range from the recent Pleistocene to late Pliocene (235,000-2.25million years ago). The Pacific A. achilles is the most divergent (and possibly ancestral) species with mtDNA dcorr≈0.04, whereas the other two Pacific species (A. japonicus and A. nigricans) are distinguishable only at a population or subspecies level (ΦST=0.6533, P<0.001). Little population structure was observed within species, with evidence of recent population expansion across all four geographic ranges. We detected sharing of mtDNA haplotypes between species and extensive hybridization based on microsatellites, consistent with later generation hybrids but also the effects of allele homoplasy. Despite extensive introgression, 98% of specimens had concordance between mtDNA lineage and species identification based on external morphology, indicating that species integrity may not be eroding. The A. nigricans complex demonstrates a range of outcomes from incomplete speciation to secondary contact to decreasing hybridization with increasing evolutionary depth.