Molecular phylogeny and historical biogeography of the Indonesian freshwater fish Rasbora lateristriata species complex (Actinopterygii: Cyprinidae)

Indonesia is one of megadiverse countries that hold a huge number of world’s biodiversity. Indonesia is a home for more than 1200 species of freshwater fish. Owing to human activities, this biodiversity is under serious threats. Due to their inability to disperse through non-freshwater environments, freshwater fish are highly vulnerable to pollution and environmental changes. Basic scientific information on, e.g., taxonomy, phylogeny, ecology, and genetic diversity should be urgently collected before they disappear. Small freshwater fishes in genus Rasbora (87 valid species) are distributed in a large geographical area, ranging from western India to Lesser Sunda Islands of Indonesia, as far as Lombok and Sumbawa Islands. Among them, 66 species occur in Indonesia. Rasbora lateristriata was described from Java Island but its taxonomy, phylogeny, and distributional boundary have not been fully studied. This species occur in both western (Java and Bali Islands) and eastern (Lombok and Sumbawa Islands) sides of Wallace’s Line, a geographical barrier between Indomalaya Ecozone in the west with fauna of the Asian origin and Australasia Ecozone in the east with those of the Australian origin. When and how this species crossed Wallace’s Line is one of the biggest enigmas in the historical biogeography of this region. Rasbora baliensis was described as a species endemic to Balinese lakes but its taxonomic status has been controversial in relation to R. lateristriata. I collected rasbora samples from 47 freshwater localities of Java and five neighboring Islands, which included 236 individuals assignable to R. lateristriata or R. baliensis. I extracted DNA from several individuals representing each locality, amplified a part of 4 genes (mitochondrial COI and Cytb genes and nuclear RAG1 and opsin genes), and sequenced them. These genes were also sequenced for a representative individual of other Rasbora species that I collected. Molecular phylogenetic analyses were conducted with these DNA sequences and those obtained from public databases for additional species. I also conducted morphological analyses with many meristic and morphometric characters, including ones that were used to describe R. baliensis by Brittan (1954). Molecular analyses using four genes, as well as morphological analyses featuring the body color pattern consistently supported that the currently recognized R. lateristriata forms a species complex including at least four major lineages that possibly represent different species. In one of the major lineages, Balinese individuals clustered tightly with those from East Javanese, Lombok and Sumbawa localities, calling for taxonomic revision on R. baliensis. The other three major lineages occur in distinct regions of central, west-central, and western Java and they can be clearly discriminated by the combination of melanophore pigment patterns in the basicaudal blotch and the supra anal pigment. Molecular phylogeny of this study suggested west-to-east divergences of the R. lateristriata species complex. R. lateristriata likely had an origin in Sumatra or western parts of Java and then migrated to the east before it crossed Wallace’s Line, colonizing Lombok and Sumbawa Islands. Based on the relaxed-clock Bayesian estimation of divergence times using the nuclear gene sequences, the divergences of this species complex in Java Island probably occurred from the late Miocene to Plio-Pleistocene. The dispersal over Wallace’s Line occurred very recently (less than five hundred thousand years ago) either naturally or by human introduction. In conclusion, the present study revealed some hidden biodiversity on Rasbora fish in Java, provided new molecular and morphological evidence to revise the taxonomy of R. lateristriata and R. baliensis, and proposed a new hypothesis on the origin and migrational pathway of the R. lateristriata species complex. As their natural habitats are rapidly deteriorated by human activities, many freshwater fish species other than the rasboras await molecular and morphological investigations. As demonstrated in this study, multidisciplinary approaches by field sampling, morphological investigations, molecular experiments, and computational analyses will be effective to tackle complex evolutionary issues and provide basic scientific knowledge necessary to design conservation plans on Indonesian fauna. Glossary of specialized terms Bayesian method: a method using Bayesian principle for estimating posterior probabilities of phylogenetic trees based on observed molecular data under a particular model of sequence change and its optimized parameters. Markov chain Monte Carlo (MCMC) process is usually employed to heuristically search for better trees in multi-dimensional space of trees and parameters. MrBayes is a program for phylogenetic inference which performs the Bayesian analysis Biodiversity (Biological diversity): variability among living organisms, including plants, animals and microbes. The biodiversity includes species diversity, genetic diversity within species, and ecosystem diversity Bootstrap: a procedure to assess the precision in estimating the phylogenetic tree by resampling a random subset of the original data matrix (DNA sequences) Clade (monophyletic group): a group of organisms (taxa) that include a common ancestor and all descendants of that ancestor Conspecific: an organism belonging to the same species Cryptic species: two or more species with very similar morphological appearance (and thus classified into a single species conventionally) but are genetically distinct in the species level Haplotype (haploid genotype): a group of genes within a chromosome of an organism which is inherited from a single parent. Mitochondrial DNA genotype is sometimes called a haplotype because mtDNA inherits maternally only from a mother to her children Historical biogeography: a study of species distributions in a geographical region through the geological time, elucidated by the phylogenetic study and distribution information Lineage sorting: the random process of fixation of gene lineages along a species lineage Maximum likelihood: the maximum likelihood method in phylogenetic analyses infers the maximum likelihood tree that has the highest probability of realizing observed molecular data under a particular model of sequence change and its parameters. One of the popular softwares to perform phylogenetic inference based on the maximum likelihood criterion is GARLI (Genetic Algorithm for Rapid Likelihood Inference) MEGA (Molecular Evolutionary Genetic Analysis): a software package for conducting various tasks in molecular evolutionary studies, e.g., sequence alignment, inferring phylogenetic trees, estimating rates of molecular evolution, calculating genetic distances and testing evolutionary hypotheses Meristic: countable traits that can be used for identifying or describing a species, e.g., number of fins, number of scales or number of gills Morphometric: measurable or quantitative traits, e.g., size, distance or proportion in morphological characters Node: a connecting point in the phylogenetic tree, which represents a common ancestor of descendants Outgroup: an outgroup in phylogenetic analyses is a group of taxa (genes or organisms) that can be used as a reference to determine the root position of ingroup taxa and infer the evolutionary direction of character changes (i.e., ancestral vs. derived characters) Phylogeny (phylogenetic tree): is a diagrammatic hypothesis about the evolutionary relationships of a group of organisms or genes Phylogeography: a study concerned with the principles and processes governing the geographic distributions of genealogical lineages, especially those within and among closely related species Polymerase Chain Reaction (PCR): a technique in molecular biology to amplify a segment of DNA into multiple copies Quaternary glaciation: a cooling event in Quaternary period when the earth experienced extreme cooling and the ice sheet was expanded globally. The Quaternary glacial maxima appeared periodically from 2.58 million years ago to 11,000 years ago Species complex: a group of closely related species with very similar morphological appearance, so that the species delimitation between them is obscure Sympatric: a distributional state of multiple organisms occurring within a locality or an area Taxon (taxa in plural): a taxonomic group or unit, a group of one or more populations of an organism or organisms seen by taxonomists to form a unit Topology: a branching pattern of a phylogenetic tree Type locality: a location where the designated type specimen was originally collected Type specimen: is particular organismal individuals to which the scientific name of a species was permanently attached according to the description based on these specimens. Holotype is a main specimen which is designated in the original description of a species by the original author. Paratype refers to several additional specimens designated together with the holotype. Usually, a taxonomist describes new species by observing several specimens. One specimen is assigned to the holotype and the rest is designated as the paratype Wallace’s Line: is a hypothetical faunal boundary which separates Asian and Australasian faunas with different origins. Wallace’s Line runs through the Lombok Strait between Bali and Lombok Islands and through the Makassar Strait between Borneo and Sulawesi Islands in Indonesia