﻿Tectariadanangensis (Tectariaceae), a new fern species from Vietnam

﻿Abstract A new fern species, Tectariadanangensis (Tectariaceae) from Vietnam, which had long been misreported as T.crenata, is described and illustrated. The new species resembles T.poilanei, a species long neglected in the fern flora of Indochina, in the frond shape and sori arrangement, but differs by its irregularly 2-rowed sori (versus regularly 2-rowed, distantly and evenly arranged) between lateral veins of pinnae, fronds being more or less dimorphic (versus monomorphic) and basal pinnae each with a base-joined (versus free) lobe. Phylogenetic analyses of five plastid regions (atpB, ndhF + ndhF-trnN, rbcL, rps16-matK + matK and trnL-F) suggested T.danangensis has a close affinity to T.harlandii. Tectariadanangensis appears to be an intermediate species between T.harlandii and T.poilanei.


Introduction
The fern species Tectaria crenata Cav. represents a morphologically distinctive group in the genus Tectaria Cav. (Tectariaceae). It was originally described on the basis of plants from the Mariana Islands and is characterised by the 1-pinnate fronds and indusiate sori in regular rows parallel to lateral veins of pinnae (Copeland 1907). Tardieu-Blot and Christensen (1941) recorded T. crenata in the flora of Indochina and cited five collections from Vietnam. However, T. crenata was reported by Holttum (1991) only from western Malesia, Philippines and southern Pacific Islands, but not in Indochina (including Cambodia, Laos, southern Myanmar, Thailand and Vietnam). The distribution of T. crenata in Vietnam remains uncertain (Hassler 2004(Hassler -2021. During recent years, we examined herbarium specimens of Tectaria from Asia in many herbaria and did not find any specimens of T. crenata from Indochina (excluding Peninsular Thailand) with typical morphology of this species as those in Malesia and Pacific Islands. The specimens from Vietnam, cited as T. crenata by Tardieu-Blot and Christensen (1941), turned out to represent an undescribed species which is reported here as T. danangensis. To test the relationships of T. danangensis with other species, we also conducted phylogenetic analyses of sequences of five plastid regions (atpB, ndhF + ndhF-trnN, rbcL, rps16-matK + matK and trnL-F).

Methods
For morphological comparisons, we studied herbarium specimens from Indochina in Herbaria BM, BO, CDBI, E, HN, HNU, IBSC, K, KUN, L, P, PE, SING and TAIF. We also conducted field observations of Tectaria species in Vietnam focusing on the variations of frond dimorphism, the shape and number of lateral pinnae, venation, sori arrangement and the presence or absence of indusia.
To infer the phylogenetic position of T. danangensis, we assembled a sequence matrix containing five plastid regions (atpB, ndhF + ndhF-trnN, rbcL, rps16-matK + matK and trnL-F) of 61 specimens (Appendix 1). The sampling was based on previous phylogenetic studies of Tectaria by Ding et al. (2014), Zhang et al. (2017) and Dong et al. (2018). Tectaria crenata was revealed to be a non-monophyletic species, but its sampled specimens from western Malesia to the Solomon Islands were resolved in a strongly supported clade with T. decurrens (C. Presl) Copel. and T. sulitii Copel. (Dong et al., in press). One of the analysed specimens, Chen et al. SITW11094 (BSIP, IBSC, TNM), was used here to represent T. crenata. Except for one specimen of T. danangensis (i.e. Dong 4909) which was newly sequenced and analysed in this study, other specimens were analysed in previous studies and their corresponding sequences are available in GenBank. The methods to obtain and align the five cpDNA sequences for Dong 4909 have been as described in Ding et al. (2014).
We analysed the matrix using Bayesian Inference (BI), Maximum Likelihood (ML) and Maximum Parsimony (MP). The MP analysis was conducted in PAUP* version 4.0d100 (Swofford 2002), with all characters weighted equally and gaps treated as missing data. One thousand heuristic replicated searches were carried out using random stepwise addition with branch swapping by tree bisection-reconnection (TBR), saving 100 trees per replicate. Bootstrap values (BS) were calculated with 1000 heuristic bootstrap replicates, one random sequence addition and TBR swapping. For BI and ML analyses, we used the software jModelTest (Posada 2008) to determine the best-fitting substitution models for the concatenated sequences and the results suggested GTR+G+I as the best-fitting model. The BI analysis was conducted with MrBayes 3.2.6 (Ronquist et al. 2012), using 10 million generations with one tree sampled every 1,000 generations; four runs with four chains were performed in parallel. The first 25% trees were discarded as burn-in. The ML analysis was conducted using raxmlGUI 2.0 (Edler et al. 2020). A thorough tree search for the best ML tree was performed. The ML bootstrap analysis was performed with 1000 replications. The analysed sequence matrix and resulting trees are available in Dryad Digital Repository (https:// doi.org/10.5061/dryad.51c59zw9t).

Results
Morphological comparisons showed that the specimens recorded as T. crenata by Tardieu-Blot and Christensen (1941) represent an undescribed species which is recognised as T. danangensis. This new species superficially resembles T. crenata in the 1-pinnate fronds and entire pinnae, but distinctly differs in sori features, such as being borne on anastomosing veins (versus terminal on free veins included in areoles) and in irregular two rows (versus regularly 2-rowed, distantly and evenly arranged) between lateral veins of pinnae/segments ( Fig. 1A and B). Based on herbarium specimens and recent collections, we found that T. danangensis is quite variable in the frond dimorphism, with fertile fronds contracted to different extents compared with sterile ones and its sori are in irregular two rows between lateral veins, close or distant to each other. A few specimens of T. danangensis with less contracted fertile fronds are similar to those of T. poilanei Tardieu, but differ mainly in their irregular 2-rowed sori (versus regularly 2-rowed) between lateral veins, upper pinnae mostly being adnate (versus pointed) to rachis and basal pinnae each having a basiscopic base-joined (versus free) lobe (Figs 1 and 2). We detected a total of 25 herbarium collections of T. danangensis containing fertile fronds, of which seven collections bore evidently abortive sporangia.
Our phylogenetic analyses of cpDNA sequences with all three methods (BI, ML or MP) consistently resolved T. danangensis in Clade IV-8 of Tectaria (Fig. 3). Based on the current sampling, two specimens of T. danangensis and an unidentified specimen (Zhang et al. 8817, for which we had no chance to examine the morphology) formed a strongly support sister relationship with T. harlandii clade including T. × hongkongensis S.Y. Dong and a T. harlandiii-like specimen (PP = 1.0, MLBS = 94% and MPBL = 90%). In contrast, T. crenata and allied species were resolved in a different clade (IV-9, Fig. 3). Though T. danangensis was suggested as having a close affinity to T. harlandii (Hook.) C.M. Kuo, these two species are morphologically strikingly different in sori features. Specifically, T. danangensis has discrete sori, whereas T. harlandii has nearly acrostichoid sori. A comparison of morphological characters amongst T. danangensis, T. poilanei and T. harlandii is listed in Table 1.

Discussion
Tardieu-Blot and Christensen (1941) overlooked the sori differences between T. danangensis and T. crenata and misidentified the former as the latter in Vietnam. Though having similar shape and dissection of fronds to T. danangensis, T. crenata and its allied species in Clade IV-9 (Fig. 3) (including T. decurrens, T. pleiosora (Alderw.) C. Chr. and T. repanda (Willd.) Holttum) differ from T. danangensis in their characteristic sori which are large and regularly 2-rowed between lateral veins, with each sorus being terminal on a single veinlet in an areole (Tagawa and Iwatsuki 1988: 372;Holttum 1991: 80). Such sori features are stable in these species and can be considered as a synapomorphy for Clade IV-9. In contrast, for species in Clade IV-8, the sori are never in regular two rows between lateral veins nor terminal on free veinlets included in areoles. Instead, their sori are relatively small, scattered between lateral veins and mostly borne on anastomosing veins in most species clustered in Clade IV-8, except for T. danangensis, T. harlandii and T. × hongkongensis. Tectaria danangensis has a unique arrangement of sori which are in irregular two rows between lateral veins ( Fig. 1A and B); while in T. harlandii and T. × hongkongensis, the sori are nearly acrostichoid, with sporangia running along veinlets between lateral veins, as shown in Zhao and Dong (2016: Fig. 2C).
By examining specimens of all Tectaria species with 1-pinnate, pinnae-entire fronds recorded in Indochina and nearby regions (Tardieu-Blot and Christensen 1941; Tagawa and Iwatsuki 1988;Xing et al. 2013;Fraser-Jenkins et al. 2018), we found that some specimens of T. danangensis look very like those of T. fissa (Kunze) Holttum, a species frequently occurring in western Malesia but not in Indochina (Holttum 1991;Lindsay and Middleton 2012 onwards). A detailed comparison (Table 1) showed that T. danangensis differs from T. fissa and other species having 1-pinnate fronds by its venation lacking distinct transverse veins between lateral veins and its sori being generally in only two rows (versus 4-6 rows) between lateral veins. Tectaria danangensis appears to be an intermediate species between T. harlandii and T. poilanei; the latter (T. poilanei) has long been neglected in literature accounting for the fern flora of Indochina (e.g. Tardieu-Blot and Christensen 1941; Tagawa and Iwatsuki 1988;Phan 2010;Lindsay and Middleton 2012 onwards). According to herbarium specimens examined, T. danangensis is not rare in Vietnam; it has been collected from 1837 to 2014 across nearly all the country and, morphologically, is quite variable in the frond dimorphism and sori distribution between lateral veins. As shown in Table 1, some characters in T. danangensis, such as frond dimorphism, attachment pattern of pinnae to rachis, venation and sori distribution, exhibit intermediate states of those between T. harlandii and T. poilanei. Notably, T. poilanei is quite stable in pinnae features (i.e. the broad-lanceolate shape, lower pinnae consistently being petiolulate and basal pinnae each bearing a free basiscopic lobe), venation lacking transverse veins between lateral veins and regularly 2-rowed well-spaced sori ( Fig. 1C and D); this species is currently represented, so far as we know, by its type specimen from southern Vietnam (Tardieu-Blot 1940) and a few collections from Thailand extend its distribution (e.g. Diagnosis. Tectaria danangensis is similar to T. poilanei Tardieu, but differs in its irregularly 2-rowed sori (versus regularly 2-rowed, well-spaced and evenly arranged) between lateral veins, fronds more or less being dimorphic (versus monomorphic) and basal pinnae each having a base-joined (versus free) lobe.
Additional specimens examined (