Another Fennoscandian Tortella species with fragile leaves, Tortella fragmenta (Pottiaceae, Bryophyta)

The well-known limestone species Tortella fragilis consists of two species, which are clearly circumscribed by the nuclear molecular marker ITS, and the plastid atpB-rbcL and rps4. The morphological differences between the two are relatively small. Because all specimens cannot be identified with certainty they could be understood as semi-cryptic. The two differ in the orientation of dry leaves and the appearance of papillae on lamina cells in mid-leaf. Dry leaves of T. fragilis s.str. are often screwed or slightly so, and near the shoot apex numerous leaves are sometimes tightly adhering (or parallel) to each other and together slightly screwed, or sometimes more loosely so or somewhat curled, and the lamina cells are densely papillose with papillae that are difficult to distinguish individually and obscure the cell walls. In T. fragmenta, which is described as new, the leaves are at most slightly screwed, curled, also near shoot apex, and not or hardly adhering to each other, and the lamina cells are moderately densely papillose with distinct and sometimes forked papillae that do not obscure the cell walls. Tortella fragilis is widespread in Fennoscandia, whereas T. fragmenta occurs primarily in the mountain range, with scattered occurrences in the lowlands of the boreal zone. Both species occur in calcareous or base-rich sites, with a tendency for T. fragilis to grow in wetter sites than T. fragmenta.

Methods
For the molecular part of this study, 24 specimens of Tortella fragilis s.l. from Fennoscandia were sequenced and sequences for two additional specimens, from Greenland and Gorno-Altai in Russia, were downloaded from GenBank. Based on Hedenäs (2015), Köckinger and Hedenäs (2017), Köckinger, et al. (2018), and (Köckinger and Hedenäs 2023), 53 specimens belonging to 15 other Tortella species, two specimens of Chionoloma tenuirostre, and two of Trichostomum crispulum Bruch were selected as outgroup.

The nuclear internal transcribed spacers 1 and 2 (ITS) and the plastid atpB-rbcL spacer (atpB-rbcL) and the rps4 gene + trnS-rps4 spacer (rps4) were studied. DNA was extraction and new sequences were generated as described by Hedenäs (2015; https://doi.org/10.1007/s00606-014-1159-9).

Nucleotide sequence fragments were edited and assembled for each DNA region using PhyDE® 0.9971 (http://www.phyde.de/index.html; accessed 7 March 2024). In the final data set, all three sequences were available for all specimens. However, the ITS sequence of D1588 was based only on the forward reading since the reverse one did not work. The assembled sequences were aligned manually in PhyDE®. Regions of partially incomplete data in the beginning and end of the sequences were identified and were excluded from subsequent analyses. Gaps were coded using the simple indel coding of Simmons and Ochoterena (2000) in SeqState (Müller, K. 2005).

广为人知的石灰岩生藓类物种脆叶扭毛藓（Tortella fragilis）实则包含两个物种，二者可通过核分子标记内转录间隔区（ITS）以及质体基因组的atpB-rbcL基因间隔区与rps4基因进行明确界定。二者的形态差异相对较小，且因所有标本均无法被确定性鉴定，故可将其视为半隐存种。二者的区别在于干燥叶片的朝向，以及叶中部叶肉细胞的乳突特征。

狭义脆叶扭毛藓（T. fragilis s.str.）的干燥叶片常呈螺旋状扭曲或轻度扭曲；在茎尖附近，多数叶片有时会紧密贴合（或平行排列）并整体轻度螺旋扭曲，有时则较为松散或略微卷曲。其叶肉细胞密布乳突，乳突个体难以分辨，且会遮蔽细胞壁。

在本次研究新定名的脆叶扭毛藓（T. fragmenta）中，叶片至多仅轻度螺旋扭曲或卷曲，即便在茎尖附近亦是如此，且叶片彼此间不贴合或几乎不贴合；其叶肉细胞乳突分布中等密度，乳突轮廓清晰，部分乳突呈分叉状，不会遮蔽细胞壁。

脆叶扭毛藓（Tortella fragilis）在芬诺斯坎底亚地区分布广泛，而新种脆叶扭毛藓（T. fragmenta）则主要分布于山地，仅在寒温带低地有零星分布。两个物种均生长于石灰岩或富碱基生境中，且脆叶扭毛藓相较于新种更倾向于栖息于湿度更高的生境。

方法
本研究的分子部分共对采自芬诺斯坎底亚的24份广义脆叶扭毛藓（Tortella fragilis s.l.）标本进行了测序，并从基因数据库（GenBank）下载了另外2份标本的序列，分别采自格陵兰与俄罗斯戈尔诺-阿尔泰地区。依据Hedenäs（2015）、Köckinger & Hedenäs（2017）、Köckinger等（2018）以及Köckinger & Hedenäs（2023）的研究，选取了隶属于15个其他扭毛藓属（Tortella）物种的53份标本、2份细喙雪藓（Chionoloma tenuirostre）标本以及2份卷叶毛口藓（Trichostomum crispulum Bruch）标本作为外类群。

本研究针对核基因组内转录间隔区1与2（ITS）、质体基因组atpB-rbcL基因间隔区（atpB-rbcL）以及rps4基因+trnS-rps4基因间隔区（rps4）开展分析。DNA提取与新序列扩增流程参照Hedenäs（2015）的方法（https://doi.org/10.1007/s00606-014-1159-9）。

本研究利用PhyDE® 0.9971软件（http://www.phyde.de/index.html；2024年3月7日访问）对每个DNA区域的核苷酸序列片段进行编辑与组装。最终数据集包含所有标本的3个目标序列，但编号为D1588的标本的ITS序列仅使用了正向测序结果，因反向测序失败。组装后的序列通过PhyDE®软件进行手动比对。序列首尾两端存在部分数据不完整的区域，已被剔除于后续分析之外。插入缺失位点采用Simmons与Ochoterena（2000）提出的简单插入缺失编码法，通过SeqState软件（Müller, K. 2005）进行编码。