Ecological divergence of cryptic species of the atyid freshwater shrimps: Caridina indistincta and Paratya australiensis species complexes at different spatial scales in South-East Queensland, Australia

Knowledge of cryptic species distributions and their relationships with environmental factors may be extremely valuable for biodiversity conservation. In freshwater ecosystems, morphologically cryptic species often have different geographic distributions that can overlap to varying extents. The importance of differential species responses to environmental conditions in determining their spatial distributions is, however, unclear. Here, we evaluated the importance of species responses to environmental drivers, in particular the physicochemical factors, in the spatial distributions and environmental relationships of two shrimp species complexes Caridina indistincta Calman, 1926 and Paratya australiensis Kemp, 1917. We analyzed shrimp specimens from 89 sites in 17 catchments across South‐East Queensland by sequencing a fragment of the mitochondrial cytochrome c oxidase subunit I gene (COI) to identify individuals. Furthermore, although there is evidence that morphologically the cryptic species of these shrimps differ very little, we made detailed morphological assessments to combine with molecular data, hoping to be able to distinguish among the species more easily and cheaply for future studies. There were significant morphological differences among the three cryptic species of the C. indistincta species complex, specifically in carapace length (cl), number of dorsal teeth (nDt), number of ventral teeth (nVt), teeth posterior to orbital margin (TPOM), and the calculated ratios A/rl, and rl/cl, while the two lineages of the P. australiensis species complex differed in the number of dorsal teeth (nDt) and the calculated ratios of A/rl, rl/cl, and rh/ch. To determine the importance of species responses in explaining the spatial distribution of cryptic species based on the species‐variables relationships, a redundancy analysis (RDA) was used to summarize these relationships. This ordination analysis showed distinct differences among cryptic species in their correlation with water quality variables and elevation. C. indistincta sp. B and C. indistincta sp. D were significantly associated with elevation and dissolved oxygen range, respectively. As well, P. australiensis lineages 4 and 6 were significantly correlated with elevation and conductivity, respectively. Overall, our results demonstrated the advantage of using analyses of biotic and abiotic variables as a valid approach for defining species responses to abiotic factors in cryptic species of atyid freshwater shrimps.

Climate change will significantly affect avian biodiversity on a global scale. Increasing temperatures over the next century will lead to shifts in species distributions, alterations in the timing of breeding and migration, changes in morphology and shifts in genetic frequencies among avian populations. The global hotspots of avian diversity are found in mountainous rainforests, regions which may be difficult to access. Therefore, effective ways of monitoring rainforest bird assemblages are vital, for both ecologists and conservationists. This thesis addresses the challenge of monitoring the effects of climate change on rainforest bird assemblages. I used two methods, point counts and automated acoustic recording, to sample the rainforest birds occupying three elevational gradients in rainforests on the east coast of Australia. In doing so, I had the following aims: 1) to determine whether biodiversity data from automated acoustic recordings made using automated recording units (ARUs) was comparable to data generated using a traditional method (point counts), 2) to assess the ability of ARUs to monitor cryptic rainforest species for long time periods, 3) to identify birds that could be used as indicator species of elevation for the purpose of long-term climate change monitoring, and 4) to investigate the driving factors of bird species richness and abundance along elevational gradients in Australian rainforests. Existing studies showed contrasting results when comparing the effectiveness of traditional avian sampling methodologies with ARUs. To address this in an Australian rainforest context, we collected data on the birds of Eungella National Park in central Queensland over two sampling periods. We found that data from point counts and ARUs was broadly similar. On average, point counts detected more species than recordings of the same duration. The respective strengths and weaknesses of point counts and ARUs are complementary, and they should be used simultaneously in future biodiversity surveys. ARUs can sample remotely, simultaneously, and for long time periods. Using ARUs, we collected a year’s worth of data on two cryptic species inhabiting rainforest in north-eastern New South Wales. Bassian Thrush Zoothera lunulata and Russet-tailed Thrush Z. heinei are secretive inhabitants of wet forests on the eastern coast of Australia. We found that the two species had differential elevational preferences: Bassian Thrush preferred elevations above 900m asl, and Russet-tailed Thrush preferred elevations below 700m asl. Recordings of song indicated that Russet-tailed Thrush bred earlier than Bassian Thrush in 2015. This, along with the elevational preferences of the two species, may be related to temperature. The use of ARUs enabled us to quantify the elevational preferences and likely breeding times of these cryptic species. Populations of Bassian Thrush in north-eastern New South Wales and south-eastern Queensland are likely to decline with increasing temperatures. Upwards shifts in the elevational ranges of rainforest birds are expected due to increasing global temperatures. Identifying the current elevational distributions of indicator species has been suggested as one way of monitoring such upwards shifts. Previous research in our study region had identified indicator species among various invertebrate and plant taxa, but information on vertebrate indicators was lacking. Using data on the elevational preferences of birds collected over one year, we identified avian indicators of lowland and highland rainforest sites in north-eastern New South Wales. These indicators may be used to detect future shifts in species elevational preferences in the region. Previous research in tropical rainforests of the Wet Tropics identified temperature as an important driver of bird species’ distributions. There was a comparative lack of information for the subtropical rainforests of north-eastern New South Wales. Our data from elevational gradients in this region indicated that temperature was significantly positively correlated with both avian species richness and abundance. Species richness declined with elevation; there was no consistent elevational pattern in abundance. We found that species’ functional traits mediated their responses to the changes in environmental conditions along the gradient: large-bodied and small-bodied species are likely to be affected in different ways by increasing temperatures. My research has determined effective ways of monitoring the effects of climate change on rainforest bird assemblages. In doing so, I have also addressed major gaps in the knowledge of two relatively understudied biodiversity hotspots on the eastern coast of Australia. The baseline data presented in this thesis allows future researchers to detect changes in the avian biodiversity of the study regions, and represents a significant contribution to ornithology and climate change research in Australia and internationally.

Although widely recognized as essential actors of ecosystem processes and representing a significant part of the Earth's biodiversity, free-living protists are poorly known. Major questions concerning their diversity, ecology, and evolution remain completely unresolved, partly because our knowledge of their taxonomy, at each different levels, is still extremely limited. In this thesis, I used testate amoebae as model organisms to get insights into the diversity and biogeography of protists. Testate amoebae are a polyphyletic group of free-living protists that are characterized by a shell composed of proteinaceous, calcareous, or siliceous material. The shells are differentiated by their shapes and ultrastructures, allowing for the identification of morphospecies more easily than in the vast majority of other protists. However, the taxonomic validity of the characters used to define morphospecies and the true diversity of testate amoebae are largely unknown. We studied the taxonomy of cyphoderiid testate amoebae (Rhizaria: Euglyphida), by combining morphological and molecular genetic approaches (chapters 2 and 3). We used light- and scanning electron microscopy to analyze the shell ultrastructure and biometry of several Cyphoderia morphospecies and sequenced gene portions of the Cytochrome Oxidase Subunit 1 (COI) and nuclear small subunit rDNA (SSU). These analyses revealed that while several described species are morphologically distinct and well-supported by the genetic data, other species, such as Cyphoderia ampulla, represent a complex of cryptic or pseudo-cryptic species. The presence of cryptic or pseudo-cryptic Cyphoderia species illustrates the limits of a morphology-based approach for assessing protist diversity using only characters visible by light microscopy. DNA sequence analyses may allow for a better resolution for both species identification and diversity estimates, provided that appropriate genetic markers can be developed. Genetic marker development in protists is particularly challenging because their representation in the international genomic databases is still very limited. The SSU marker also allowed us to build an extensive phylogeny of eulgyhid testate amoebae. We then used this phylogeny as a framework to analyze macro-evolutionary processes in testate amoebae, in particular, how easily a species can cross an ecological barrier and colonize new habitats. The taxa included in our phylogeny occurred either in marine, marine supralittoral or freshwater habitats, and species of each habitat segregated into distinct sub-clades. This indicates that transitions between these environments occur only rarely in the course of evolution (chapter 4). A reliable taxonomy for microorganisms is not only an essential prerequisite for estimating species diversities, it would also be a key component to resolve the long-lasting debate of whether free-living microorganisms are strictly cosmopolitan, or whether at least some species have limited geographic distributions (chapter 1). We analyzed Arcellinida and Euglyphida testate amoeba communities from Amsterdam Island, one of the most remote islands in the world located in the Indian Ocean, to search for endemic species with limited geographic distributions (chapter 5). In macroorganisms, the degree of endemism is generally high in such remote islands. Our extensive inventory of testate amoeba morphospecies provided no clear evidence for endemism. However, given that the morphology-based species identification gives only a limited taxonomic resolution, we cannot exclude the presence of morphologically non-distinctive endemic species (cryptic species). Thus, such taxonomic uncertainties undermine biogeographical studies of testate amoebae and other protists. To circumvent taxonomical uncertainties in biogeographical studies, we analyzed the distribution of a morphologically highly distinctive testate amoeba, Nebela ansata (chapter 6). We report the presence of this species in Nova Scotia, Canada and at sites in New Jersey where it was initially described. An extensive literature survey further confirmed the absence of this species from both, the remaining North American continent as well as other continents. Together, these data provide an unusually convincing case of a free-living microorganism with a very limited distribution range along the temperate East American coast. Overall, my thesis illustrates how, in most cases, the current morphology-based species identification greatly underestimates testate amoeba diversity and calls for an improvement of the taxonomic resolution and reliability. The extension of the current taxonomy using molecular-genetic tools should greatly contribute to this task and also shed light on the debate on the distribution of protists and other microorganisms. Together, such studies will provide a first step towards a better understanding of the diversity and evolution of microorganisms and improve their utility as model organisms in both fundamental and applied questions.

In recent decades, the use of molecular techniques in rotifers has revealed the existence of many cryptic species. Although strong competition is expected among cryptic species, these species are often sympatric. Here, we present a review of sympatric cryptic rotifer species, focusing on those cases in which niche differentiation has been investigated. There are at least 42 cryptic rotifer species complexes, and species coexistence is commonly reported. Ecological differentiation among cryptic species has been detected in several complexes. However, the only available information regarding mechanisms that allow cryptic species coexistence is for several species of the Brachionus plicatilis complex: B. plicatilis, B. ibericus, B. rotundiformis and B. manjavacas. According to these studies, when species differ in body size, niche differentiation is related to abiotic and biotic factors (e.g. the differential use of resources and vulnerability to predation). In contrast, if species are almost identical in body size, their biotic niches and competitive abilities are very similar, and niche differentiation is facilitated by the differences in the species responses to fluctuating, physical environment in combination with the divergence in life-history traits related to diapause. Further studies of additional cryptic rotifer species are essential to know the generality of these conclusions.

Freshwater fishes often exhibit high genetic population structure due to the prevalence of dispersal barriers (e.g., waterfalls) whereas population structure in diadromous fishes tends to be weaker and driven by natal homing behaviour and/or isolation by distance. The Australian smelt (Retropinnidae: Retropinna semoni) is a native fish with a broad distribution spanning inland and coastal drainages of south-eastern Australia. Previous studies have demonstrated variability in population genetic structure and movement behaviour (potamodromy, facultative diadromy, estuarine residence) across the southern part of its geographic range. Some of this variability may be explained by the existence of multiple cryptic species. Here, we examined genetic structure of populations towards the northern extent of the species’ distribution, using ten microsatellite loci and sequences of the mitochondrial cyt b gene. We tested the hypothesis that genetic connectivity among rivers should be low due to a lack of dispersal via the marine environment, but high within rivers due to dispersal. We investigated populations corresponding with two putative cryptic species, SEQ-North (SEQ-N), and SEQ-South (SEQ-S) lineages occurring in south east Queensland drainages. These two groups formed monophyletic clades in the mtDNA gene tree and among river phylogeographic structure was also evident within each clade. In agreement with our hypothesis, highly significant overall FST values suggested that both groups exhibit very low dispersal among rivers (SEQ-S FST = 0.13; SEQ-N FST= 0.27). Microsatellite data indicated that connectivity among sites within rivers was also limited, suggesting dispersal may not homogenise populations at the within-river scale. Northern groups in the Australian smelt cryptic species complex exhibit comparatively higher among-river population structure and smaller geographic ranges than southern groups. These properties make northern Australian smelt populations potentially susceptible to future conservation threats, and we define eight genetically distinct management units along south east Queensland to guide future conservation management. The present findings at least can assist managers to plan for effective conservation and management of different fish species along coastal drainages of south east Queensland, Australia.

Sulawesi is one of the four Greater Sunda Islands characterized by rivers and lakes habitats of freshwater shrimps and prawns with a high degree of endemism. The freshwater shrimp and prawns diversity in the Batusuya river of Donggala is less studied compared to the ancient lakes in the Central Sulawesi area. This study aimed to determine the diversity of freshwater shrimp and prawns of the Batusuya river. The sample was collected in November 2016 using tray net. The study site divided into three sampling stations based on the type of habitat. Nine species of freshwater shrimps and prawns were found, i.e., Macrobrachium australe , M. esculentum , M. horstii , M. lar , M. placidulum , Caridina brevicarpalis , C. gracilipes , C. weberi , and Atyopsis spinipes . Six species were found in all localities including Macrobrachium australe , M. esculentum , M. horstii , M. lar , C. brevicarpalis , and C. gracilipes , while M. placidulum , C. weberi , and A. spinipes were not found in all stations. The highest diversity index (H') was found in the station III indicated freshwater shrimps and prawns favored the environmental conditions of this station. The highest measure of species richness (R) was found in the station I and presumably related to the life cycle of freshwater shrimps and prawns which is amphidromous. The occurrence of nine species of freshwater shrimps and prawns along the Batusuya river indicated the environmental condition of the stream was suitable for most freshwater shrimps and prawns, and the Macrobrachium australe was found as the most abundant species.

Freshwater fishes often exhibit high genetic population structure due to the prevalence of dispersal barriers (e.g., waterfalls) whereas population structure in diadromous fishes tends to be weaker and driven by natal homing behaviour and/or isolation by distance. The Australian smelt (Retropinninae: Retropinna semoni) is a facultatively diadromous fish with a broad distribution spanning inland and coastal drainages of south-eastern Australia. Previous studies have demonstrated variability in population genetic structure and movement behaviour (potamodromy, facultative diadromy, estuarine residence) across the southern part of its geographic range. Some of this variability may be explained by the existence of multiple cryptic species. Here, we examined genetic structure of populations at the northern extent of the species’ distribution, using ten microsatellite loci and sequences of the mitochondrial cyt b gene. We tested the hypothesis that connectivity among rivers should be low due to a lack of dispersal via the marine environment, but high within rivers due to potamodromous behaviour. We investigated populations corresponding with two putative cryptic species, the South East Queensland (SEQ), and Central East Queensland (CEQ) lineages. In agreement with our hypothesis, highly significant overall FST values suggested that both groups exhibit very low dispersal among rivers (SEQ FST = 0.13; CEQ FST = 0.30). The two putative cryptic species, formed monophyletic clades in the mtDNA gene tree and among river phylogeographic structure was also evident within clades. Microsatellite data indicated that connectivity among sites within rivers was also limited, suggesting potamodromous behaviour does not homogenise populations at the within-river scale. Overall, northern groups in the smelt cryptic species exhibit higher among-river population structure and smaller geographic ranges than southern groups. These properties make northern Australian smelt populations potentially susceptible to future conservation threats, and we define eight genetically distinct management units to guide future conservation management.

Species complexes within epiphytic diatoms and their relevance for the bioindication of trophic status

Sexual traits that promote species recognition are important drivers of reproductive isolation, especially among closely related species. Identifying neural processes that shape species differences in recognition is crucial for understanding the causal mechanisms of reproductive isolation. Temporal patterns are salient features of sexual signals that are widely used in species recognition by several taxa, including anurans. Recent advances in our understanding of temporal processing by the anuran auditory system provide an opportunity to investigate the neural basis of species-specific recognition. The anuran inferior colliculus consists of neurons that are selective for temporal features of calls. Of potential relevance are auditory neurons known as interval-counting neurons (ICNs) that are often selective for the pulse rate of conspecific advertisement calls. Here, we tested the hypothesis that ICNs mediate acoustic species recognition by exploiting the known differences in temporal selectivity in two cryptic species of gray treefrog (Hyla chrysoscelis and Hyla versicolor). We examined the extent to which the threshold number of pulses required to elicit behavioral responses from females and neural responses from ICNs was similar within each species but potentially different between the two species. In support of our hypothesis, we found that a species difference in behavioral pulse number thresholds closely matched the species difference in neural pulse number thresholds. However, this relationship held only for ICNs that exhibited band-pass tuning for conspecific pulse rates. Together, these findings suggest that differences in temporal processing of a subset of ICNs provide a mechanistic explanation for reproductive isolation between two cryptic treefrog species.

Most of gall-inducers are monophagus or oligophagus, which utilize a narrow spectrum of host plants and organs. Diversity of gall inducers and the specific relationship between galling insects and host plants are considered as a consequence of adaptive radiation. Galls could be considered as extension of phenotypes of the gall indusers. This study focused on the relationship between Daphnephila spp. (Diptera: Cecidomyiidae) and Machilus (Lauraceae) in Taiwan based on morphological characters of insect and galls, molecular, and biological data in order to clarify the galler taxonomy and the phylogenetic relationship. After examining all the plant species of Machilus in Taiwan, the acquired galls were sorted into 38 morphological species based on their gall shape, galling position, and host plant information. Galls with similar shaped could be further groups into 8 series, i.e., spindle-shaped, needle-like, urn-shaped, little urn-shaped, obovate, club-shaped, bird-head-shaped and ovoid gall serieses. There are significant differences among gall seires but smaller variation within each gall series. Molecular analansis based on gene revealed that most of gall midges with similar shapes (gall morphospecies) are monophyletic group. Clear differentiation ranging from 1.3% to 14.3% exists between each group and only a few less than 2% which is mainly belong to club-shaped series. Morphological differences were found between midges from stem and leaf galls, espcieally on larval spatula and adult genitalia. Only trievial differences could be found for the midges within stem or leaf galling groups. The overall evdiences suggest that there are at least 9 species in Daphnephila, including 5 named species (Daphnephila sueyenae, D. ornithocephaia, D. stenocalia, D. taiwanensis, D. truncicola), and 4 new species. The stem gall midges inculed 1 named species (D. truncicola) and 3 unnamed species. The taxonomy of leaf gall midges are more complicated. The mouse-like gall series is a species complex, which contain D. taiwanensis and a cryptic species. The club-shaped, bird-head-like, and the urned-shaped series formed another species complex, including two named species (D. stenocalia and D. ornithocephala) and a new species (urned-shaped gall inducer). The midges of little urn-shaped gall series are the same species of midges belonging to obovate gall series (D. sueyenae). Field observation on midges indicates the clear synchronization between reproduction of adults and phenology of host plants. Females may secrete pheromone to mate with males and the galling position and patter are determined by females. To sum up, the gall shape series (except for little urn-shaped gall series) reflect the taxonomic status at species category of Daphnephila gall midges, whetheras the variability with gall shape series may not always reflect the species status. It is necessary to obtain eveidences from differet aspects and to integrate the overall variation for making correct taxonomic determination for the gall midges.

Sympatric distribution and temporal overlap of cryptic zooplankton species pose a challenge to the framework of the niche differentiation theory and the mechanisms underlying the coexistence of potentially strong competitors. In this study, we collected Brachionus calyciflorus Pallas monthly from Lake Tingtang over the course of 1 year and clonally cultured them in the laboratory. Finally, 204 clones were established, and their nuDNA ITS1 and mtCOI sequences were analyzed. The phylogenetic relationships and GMYC model analyses revealed that B. calyciflorus was a species complex composed of three cryptic species. Cryptic species BcI-W, and BcII-S and Bc-SW underwent a clear seasonal succession. However, BcII-S and Bc-SW overlapped in June and July, and all the three cryptic species coexisted in November. Different preferences to water temperature might contribute to the seasonal succession. More investment in sexual reproduction accelerated the disappearance of cryptic species from the water column during the seasonal succession process. Differential responses in rate of population growth of the two overlapping cryptic species to increasing food level might reduce the intensity of competition between them and promote their coexistence over time. Differences in population growth rate partially resulted in the different relative abundances of the two overlapping cryptic species.

In this study, species boundaries were examined for 15 described and 2 undescribed species within the economically important Culicoides subg. Avaritia Fox from Australasia and Eastern Asia. We used an integrative taxonomic approach incorporating DNA barcoding, nuclear gene sequencing, and retrospective morphological analyses. Some arbovirus vector species such as Culicoides fulvus Sen and Das Gupta and Culicoides wadai Kitaoka were genetically and morphologically uniform across sampled distributions, but others including Culicoides actoni Smith and Culicoides brevipalpis Delfinado contained 2 or more genetically independent populations of 'cryptic species' that in some cases were sympatric. Some of these 'cryptic species' exhibited consistent morphological differences, while differences are yet to be found for others species. Additionally, an undescribed species, C. Avaritia sp. No. 3, was found to be synonymous with C. fulvus. These results refine our understanding of the distribution of individual species of C. subg. Avaritia and demonstrate that species descriptions and distribution records need revision for part of the Culicoides fauna. Furthermore, because vector competence studies for most of these species are based entirely on Australian populations, the competence of the putative cryptic species identified elsewhere will require independent assessment. Finally, integrative taxonomic assessment requires genetic and morphological assessment of material from the type localities in order to clarify the status and distribution of species, especially for clades containing cryptic species. International collaboration is needed to facilitate this research.

The spatial patterns of genetic structure among juveniles of long-lived species can reveal the extent of interannual and geographic variation in realized larval dispersal as well as the processes that determine ecologically relevant patterns of population connectivity. However, few studies examine this temporal and spatial variation over large portions of a species' geographic range or between cryptic species that overlap in their range. Despite the potential for long-dis- tance dispersal in blue rockfish Sebastes mystinus, a previous study of adults revealed two geo- graphically distinct, cryptic species. To determine year-to-year variation in the patterns of spatial connectivity and to elucidate the potential ecological mechanisms involved in shaping new year- classes and maintaining cryptic species, we sampled juveniles within California, USA, where both cryptic species coexist. Using microsatellite markers, we found geographic and temporal variation in the number of individuals from these distinct cryptic species within 2 new year-classes of juve- nile S. mystinus. We also found differences in the geographic patterns of genetic structure of the 2 cryptic species. Whereas one species exhibited little or no spatial genetic structure across the study region, the other exhibited a complex geographic pattern of genetic structure, with little or no genetic structure among regions, but small-scale structure within a region. The results of this study demonstrate that the spatial scales and patterns of realized dispersal of pelagic larvae vary geographically, interannually, and between closely related species with similar life-history strate- gies. Therefore, estimates of dispersal based on larval duration and patterns of adult structure need to be interpreted cautiously.

Differences in resource use or in tolerances to abiotic conditions are often invoked as potential mechanisms underlying the sympatric distribution of cryptic species. Additionally, the microbiome can provide physiological adaptations of the host to environmental conditions. We determined the intra- and interspecific variability of the microbiomes of three cryptic nematode species of the Litoditis marina species complex that co-occur, but show differences in abiotic tolerances. Roche 454 pyrosequencing of the microbial 16S rRNA gene revealed distinct bacterial communities characterized by a substantial diversity (85-513 OTUs) and many rare OTUs. The core microbiome of each species contained only very few OTUs (2-6), and four OTUs were identified as potentially generating tolerance to abiotic conditions. A controlled experiment in which nematodes from two cryptic species (Pm1 and Pm3) were fed with either an E.coli suspension or a bacterial mix was performed, and the 16S rRNA gene was sequenced using the MiSeq technology. OTU richness was 10-fold higher compared to the 454 data set and ranged between 1118 and 7864. This experiment confirmed the existence of species-specific microbiomes, a core microbiome with few OTUs, and high interindividual variability. The offered food source affected the bacterial community and illustrated different feeding behaviour between the cryptic species, with Pm3 exhibiting a higher degree of selective feeding than Pm1. Morphologically similar species belonging to the same feeding guild (bacterivores) can thus have substantial differences in their associated microbiomes and feeding strategy, which in turn may have important ramifications for biodiversity-ecosystem functioning relationships.

How many DNA markers are needed to reveal cryptic fungal species?