【目的】黑龙江省杓兰属(Cypripedium)植物表型变异丰富,为厘清物种界限模糊及亲缘关系复杂等问题,以该地区调查获取的13个杓兰属居群为材料,初步探讨其亲缘关系与分类界线。【方法】测定13个居群的18项表型性状,进行多样性分析与聚类分析;同时通过测序、组装与注释,获取其核基因ITS序列及3个叶绿体DNA片段(trnH—psbA、trnL—F与rbcL),利用DnaSP、PhyloSuite、FigTree等软件,结合NCBI数据库数据,统计核苷酸多态性并构建系统发育树。【结果】退化雄蕊(斑点与大小)可作为黑龙江杓兰属植物的关键野外识别特征。东北杓兰(C. × ventricosum)表现出显著的杂种优势,其叶片大小与株高、地径可作为分类依据。表型与系统发育分析共同表明:白花与粉花大花杓兰(C. macranthos)实为同种;杓兰(C. calceolus)与山西杓兰(C. shanxiense)可能是由同一母本分化的姐妹群;东北杓兰居群DB1、DB2、DB5聚为一支,其母本可能为杓兰,而居群DB3、DB4、DB6、DB7、DB8聚为另一支,可能源于大花杓兰,其中DB6可能与大花杓兰发生过回交;紫点杓兰(C. guttatum)则与其他种类亲缘关系较远。【结论】仅凭单一表型性状或DNA片段难以准确判定杓兰属植物的亲缘关系,尤其对于杂交种,必须综合表型、叶绿体DNA与核DNA等多层次证据,方能全面阐明其系统发育与分类界限。

【Objective】This study aims to clarify the phylo genetic relationships and taxonomic boundaries of Cypripedium in Heilongjiang Province, where the genus exhibits extensive phenotypic variation, ambiguous species delimitation, and complex phylogenetic patterns.【Method】A total of 13 natural populations collected from the region were used as the research material, we measured 18 phenotypic traits across these 13 populations and performed phenotypic diversity and cluster analyses. Furthermore, we sequenced, assembled and annotated the nrITS region and three chloroplast DNA fragments (trnH—psbA, trnL—F, and rbcL). Nucleotide polymorphism was statistically analyzed, and phylogenetic trees were reconstructed using software packages DnaSP, PhyloSuite and FigTree, incorporating relevant sequences from the NCBI public database.【Result】Staminode characteristics (presence of mottling and size) serve as key traits for field identification of C. species in Heilongjiang Province. C. × ventricosum exhibits significant heterosis, with leaf size, plant height and stem diameter at ground level providing reliable morphological markers for its classification. Integrated phenotypic and phylogenetic analyses consistently demonstrated that: the white-and pink-flowered forms of C. macranthos constitute the same species; C. calceolus and C. shanxiense likely represent sister lineages derived from the same maternal ancestor; populations DB1, DB2 and DB5 of C. × ventricosum form a distinct clade potentially originating from C. calceolus as the maternal parent, while populations DB3, DB4, DB6, DB7 and DB8 cluster in another clade likely descended from C. macranthos, with population DB6 showing evidence of possible backcrossing with C. macranthos; C. guttatum remains phylogenetically distant from all other studied species.【Conclusion】Inferring genetic relationships in Cypripedium, particularly for hybrid taxa, cannot rely solely on a single phenotypic trait or single DNA marker. A comprehensive approach integrating phenotypic data with both chloroplast and nuclear DNA sequences is essential for accurately elucidating phylogenetic relationships and taxonomic boundaries.