Limitations of Species Delimitation Based on Phylogenetic Analyses: A Case Study in the Hypogymnia hypotrypa Group (Parmeliaceae, Ascomycota)

Delimiting species boundaries among closely related lineages often requires a range of independent data sets and analytical approaches. Similar to other organismal groups, robust species circumscriptions in fungi are increasingly investigated within an empirical framework. Here we attempt to delimit species boundaries in a closely related clade of lichen-forming fungi endemic to Asia, the Hypogymnia hypotrypa group (Parmeliaceae). In the current classification, the Hypogymnia hypotrypa group includes two species: H. hypotrypa and H. flavida, which are separated based on distinctive reproductive modes, the former producing soredia but absent in the latter. We reexamined the relationship between these two species using phenotypic characters and molecular sequence data (ITS, GPD, and MCM7 sequences) to address species boundaries in this group. In addition to morphological investigations, we used Bayesian clustering to identify potential genetic groups in the H. hypotrypa/H. flavida clade. We also used a variety of empirical, sequence-based species delimitation approaches, including: the “Automatic Barcode Gap Discovery” (ABGD), the Poisson tree process model (PTP), the General Mixed Yule Coalescent (GMYC), and the multispecies coalescent approach BPP. Different species delimitation scenarios were compared using Bayes factors delimitation analysis, in addition to comparisons of pairwise genetic distances, pairwise fixation indices (FST). The majority of the species delimitation analyses implemented in this study failed to support H. hypotrypa and H. flavida as distinct lineages, as did the Bayesian clustering analysis. However, strong support for the evolutionary independence of H. hypotrypa and H. flavida was inferred using BPP and further supported by Bayes factor delimitation. In spite of rigorous morphological comparisons and a wide range of sequence-based approaches to delimit species, species boundaries in the H. hypotrypa group remain uncertain. This study reveals the potential limitations of relying on distinct reproductive strategies as diagnostic taxonomic characters for Hypogymnia and also the challenges of using popular sequence-based species delimitation methods in groups with recent diversification histories.界定近缘支系间的物种界限通常需要依托一系列独立数据集与多元分析方法。与其他生物类群相仿，真菌的稳健物种界定研究也愈发倾向于采用实证框架开展。本研究针对亚洲特有地衣型真菌（lichen-forming fungi）的一个近缘支系——裸腹叶属（Hypogymnia）hypotrypa物种群（梅衣科（Parmeliaceae））——展开物种界限界定工作。 在现行分类系统中，该H. hypotrypa物种群包含两个物种：H. hypotrypa与H. flavida，二者凭借独特的生殖模式加以区分：前者产生粉芽（soredia），后者则无该结构。我们借助表型性状与分子序列数据（内转录间隔区（Internal Transcribed Spacer, ITS）、甘油醛-3-磷酸脱氢酶（Glyceraldehyde-3-Phosphate Dehydrogenase, GPD）以及微染色体维持蛋白7基因（Minichromosome Maintenance 7, MCM7））重新审视了这两个物种间的亲缘关系，以明确该类群的物种界限。 除形态学调查外，我们采用贝叶斯聚类分析以识别H. hypotrypa与H. flavida支系中的潜在遗传类群。此外，我们还运用了多种基于序列的实证物种界定方法，包括“自动条形码间隙发现”（Automatic Barcode Gap Discovery, ABGD）、泊松树过程模型（Poisson Tree Process, PTP）、广义混合约尔-合并模型（General Mixed Yule Coalescent, GMYC）以及多物种合并分析方法BPP。我们通过贝叶斯因子界定分析对不同物种界定场景进行比较，同时辅以成对遗传距离与成对固定指数（FST）的对比分析。 本研究中实施的绝大多数物种界定分析均未支持H. hypotrypa与H. flavida为独立演化支系，贝叶斯聚类分析结果亦与之相符。不过，借助BPP分析可推断出二者具有演化独立性的强力支持，且贝叶斯因子界定分析进一步验证了这一结论。 尽管本研究开展了严谨的形态学比较与覆盖广泛的基于序列的物种界定方法，H. hypotrypa物种群的物种界限仍未明确。本研究既揭示了将独特生殖策略作为裸腹叶属（Hypogymnia）分类诊断特征的潜在局限性，也凸显了在近期分化类群中应用主流基于序列的物种界定方法所面临的挑战。