Cryptocarya kaengkrachanensis, a new species of Lauraceae from Kaeng Krachan National Park, southwest Thailand

Abstract A new species of Lauraceae, Cryptocarya kaengkrachanensis M.Z.Zhang, Yahara & Tagane, from Kaeng Krachan National Park, Phetchaburi Province, southwestern Thailand, is described and illustrated. This species is morphologically most similar to C. amygdalina in that its leaves are pinnately veined, leathery, and apparently glabrous (but microscopically hairy) abaxially, twigs are yellowish brown hairy, and fruits are 1.36 to 1.85 times longer than width. However, C. kaengkrachanensis is distinguished from C. amygdalina in having the leaves of ovate and elliptic (vs. oblong-lanceolate) with leaf aspect ratio (length:width) from 1.38 to 2.28 (vs. 2.46–3.43), and ovoid fruits (vs. ellipsoid) with stalk distinctly swollen (vs. not or only slightly swollen). In addition, phylogenetic trees constructed based on internal transcribed spacer sequences (ITS) and genome-wide SNPs using MIG-seq showed that C. kaengkrachanensis is not sister to C. amygdalina and is distinct from all the other Cryptocarya species hitherto recognized in Thailand. Analysis including other species demonstrates that C. floribunda should be a synonym of C. amygdalina, but we recognize C. scortechinii as a distinct species.


Introduction
Lauraceae, a plant family widely distributed across the world, contain an estimated 2500-3500 species in about 50 genera, and its highest species richness is found in the tropical forests of Southeast Asia and the Americas (Rohwer 1993, Li et al. 2008, Rohwer et al. 2014, Yahara et al. 2016. In Southeast Asia, trees of Lauraceae occur widely from lowlands to high elevations (van der Werff 2001, Ngernsaengsaruay et al. show that the two species from Kaeng Krachan National Park identified as "C. amygdalina" are not sister to each other in a phylogenetic tree constructed by ITS sequences and genome-wide SNPs of MIG-seq (Suyama and Matsuki 2015). By combining the molecular evidence with morphological and field observations, we revise the broader concept of "C. amygdalina" of de Kok (2015Kok ( , 2016a by recognizing three species, C. amygdalina s. str., C. scortechinii, and a taxon from Kaeng Krachen National Park.

Field observations
In Kaeng Krachan National Park (Fig. 1) Oct. 2013). All vascular plants were recorded in each plot. For trees 4 m or taller, we recorded girth and height of trunks. For trees lower than 4 m and herbs, we recorded presence/absence in each of ten 10 m × 5 m sections. For all the species distinguished in the field, we collected voucher specimens and sampled some pieces of leaves for DNA isolation (vouchers were deposited at BKF and FU). Each sample collected for DNA isolation was dried with silica gel in a zipper storage bag. In addition to plants recorded in the five plots, we also collected additional specimens with flowers or fruits from outside the plots.

DNA barcoding
For DNA extraction, we milled the dried leaf material into fine powder by QIAGEN TissueLyser and the powder was washed three times with 1 ml buffer solution (including 0.1 M HEPES, pH 8.0; 2% Mercaptoethanol; 1% PVP; 0.05 M Ascorbic acid) . DNA was then isolated from the washed powder by using the CTAB method (Doyle and Doyle 1987) with a slight modification .
We determined partial sequences of the internal transcribed spacer (ITS region) of ribosomal DNA using the following primer sets of Rohwer et al. (2009): ITS18-F (5'-GTCCACTGAACCTTATCATTTAGAGG-3') and ITS26-R (5'-GCCGT-TACTAAGGGAATCCTTGTTAG-3') and Tks Gflex DNA Polymerase (Takara Bio, Kusatsu, Japan) . The PCR reaction was carried out following the published protocols of Kress et al. (2009)   Next generation DNA sequencing -MIG-seq We amplified thousands of short sequences by using the primers of "multiplexed ISSR (inter simple sequence repeats) genotyping by sequencing" (MIG-seq, Suyama and Matsuki 2015) for 24 samples of Cryptocarya, following the protocol of Suyama and Matsuki (2015). Two steps of PCR were performed; for the 1 st PCR step, we amplified ISSR regions from genomic DNA with MIG-seq tailed ISSR primer set-1 and diluted 50 times for each 1 st PCR product with deionized water Matsuki 2015, Binh et al. 2018). The 2 nd PCR step was conducted with common and indexed primers. The 2 nd PCR products were then pooled in equimolar concentrations as a single mixture library. Fragments of size range 350-800 bp were isolated from the purified mixture of

Phylogenetic tree reconstruction
For DNA barcoding analysis, MEGA X (Kumar et al. 2018, http://www.megasoftware.net/) was used to assemble the ITS sequences of 30 samples including 29 of Cryptocarya spp. and an additional sample of Beilschmiedia sp.; MAFFT ver. 7 (http:// mafft.cbrc.jp) was used to align ITS sequences. We reconstructed a phylogenetic tree by the maximum likelihood method with Tamura 3-parameter model using MEGA X with a bootstrap test of 1500 replicates. In addition, we drew a TCS haplotype network (Clement et al. 2000) among 29 samples of Cryptocarya using POPART ver.1.7 (Leigh and Bryant 2015).
For MIG-seq, we pretreated the raw data of Cryptocarya samples following the quality control protocol of Suyama and Matsuki (2015) and Binh et al. (2018). We then assembled homologous sequences (designated as loci below) with the de novo map pipelines (ustacks, cstacks, sstacks) using Stacks ver. 1.48 (Catchen et al. 2011). First, we assembled loci by ustacks with the following settings: m = 3, M = 3, N = 2, and maximum gaps = 2 (where "m" is the minimum depth of coverage, "M" is maximum distance allowed between stacks, and "N" is the maximum distance allowed to align secondary reads to primary stacks). We then used cstacks to build a catalogue of consensus loci by assembling loci from ustacks, by setting the parameter of "number of allowed mismatches between sample (n)" as 2. Second, by using the sstacks, we associated all stacks created by ustacks with the catalog produced by cstacks. Third, we got an output vcf file containing genotypes of individuals at each locus. Subsequently, we used the vcf2phylip program (Ortiz 2019) to convert the vcf file to a phylip type file. Finally, we constructed a maximum likelihood tree with RAxML ver. 8.2 (Stamatakis 2014) and examined its reliability by bootstrapping using 1500 replicates.

Field observation
In the plot at an elevation of 360 m, we recorded three sterile trees of C. pustulata and collected a specimen (voucher specimen number T0524) from one of these trees. In the plot at 540 m, we found no trees of Cryptocarya. However, a sterile specimen of C. pustulata was collected along the roadside at 550 m (T2971). In the plot at 680 m, we recorded two sterile trees of C. pustulata for which we recorded girth × height as 110.7 cm × 25 m and 11.3 cm × 5.5 m, respectively. In addition, we collected a sterile specimen (T2195) from a tree lower than 4 m. Along the roadside at 709 m, we collected a fruiting specimen of C. amygdalina (T3090) on 30 May 2014. In the plot at 850 m, we recorded two sterile trees of Cryptocarya, both of which were lower than 4 m. However, we could not identify these trees and vouchers were not collected. In the plot at 960 m ( Fig. 1), we recorded girth × height for two trees of the Kaeng Krachan taxon as 24.3 cm × 5 m and 15.7 × 4.5 m, respectively. In addition, in the vicinity of the plot, a fruiting specimen (T2069) was collected from a tree 12 m tall on 23 Oct. 2013. Young trees of the Kaeng Krachan taxon lower than 4 m were found in all ten sections of 10 m × 5 m in the 100 m × 5 m plot at the elevation of 960 m.
The abaxial leaf blade surface of the Kaeng Krachan taxon is sparsely covered with minute hairs that are almost invisible to the naked eye or hand lens (10 ×), but visible under a microscope (25 ×). Similarly, the lower leaf surface of C. amygdalina (T3090) is sparsely covered with minute hairs that are visible only under a microscope (25 ×). Both C. amygdalina and the Kaeng Krachan taxon have scalariform to scalariformreticulate tertiary veins and it is difficult to distinguish between the two species by their venation.

Phylogenetic analysis
A phylogenetic tree constructed from ITS sequences with length of about 670 bp (Fig. 5) showed that C. amygdalina is close to C. albiramea Kosterm. (T3902) and C. pustulata, and the bootstrap support for the monophyly of the clade including these species was 84%. For the ITS sequence, two samples initially identified as C. amygdalina (T3090 collected from Kaeng Krachan, Thailand and MY479 collected from Myanmar) were identical in the ITS sequences determined. Also, two samples of C. pustulata (T2195 of Kaeng Krachan and T1545 collected from Kao Soi Dao, Chanthaburi, Thailand, the type locality) were identical but another ITS sequence of C. pustulata (T2971) differed from T2195 in one base pair (Fig.  6). On the other hand, C. amygdalina is one species in a well supported clade with the plants of the Kaeng Krachan taxon sister to this clade, differing by 10 base pairs in the ITS sequences (Fig. 6) and are distinct from each other in the ITS haplotype network (Fig. 6).
For 24 of the samples that belonged to a clade supported by 95% bootstrap value in the ITS tree (Fig. 5), we constructed a MIG-seq tree in which conspecific clusters of C. amygdalina (T3090, MY0479), the Kaeng Krachan taxon (T1883, T2069) and C. pustulata (T2195, T2971, T1545) were supported by 100% bootstrap values (Fig.  Figure 6. A haplotype network of Cryptocarya species from Thailand constructed from the ITS sequences. 7). While C. amygdalina and C. pustulata were sister to each other, the Kaeng Krachan taxon was not sister to either of these species, but instead to C. albiramea (Fig. 7), with 99% bootstrap (BS).
Based on the evidence of morphology and phylogenetic analysis presented above, the two samples (T1883 and T2069) collected from Kaeng Krachan national park clearly represent a distinct and new species, which is named as Cryptocarya kaengkrachanensis.
Other Distribution. Endemic to Thailand. The new species is currently only known in a few protected areas of Phrae, Phetchaburi and Kanchanaburi Provinces including Kaeng Krachan National Park.
Etymology. The specific epithet kaengkrachanensis is derived from the name of the national park from which the species has first been recorded.
Conservation status. Least Concern (IUCN 2012(IUCN , 2017. This species occurs in hill evergreen forests of some protected areas including Kaeng Krachan National Park and there is no sign of declining trends.

Discussion
In Kaeng Krachan National Park, we found three species of Cryptocarya that grew at different elevations. While C. pustulata was collected at lower elevations, 360 m, 550 m and 680 m, C. amygdalina and C. kaengkrachanensis were collected at higher elevations, 709 m and 960 m. Cryptocarya pustulata is a canopy tree constituent and attains a height of 25 m and we were unable to collect fertile material of this species. On the other hand, C. kaengkrachanensis is a subcanopy tree and we collected fruits from a tree 12 m tall. This species was common in the hill evergreen forest at an elevation of 960 m. Cryptocarya amygdalina and C. kaengkrachanensis are suspected to flower in different seasons because we collected a fruiting specimen of C. amygdalina (T3090) on 30 May 2014, and a fruiting specimen of C. kaengkrachanensis (T2069) on 23 Oct. 2013. The above observations in the field supported our hypothesis that there are three ecologically distinct species of Cryptocarya in Kaeng Krachan National Park.
In addition to ecological differences, the three species are genetically well differentiated. In particular, C. amygdalina and C. kaengkrachanensis differed by 10 base pairs of the ITS sequences and are placed in distant positions on both the ITS and MIG-seq trees. While C. kaengkrachanensis was sister to C. albiramea in MIG-seq tree, C. kaengkrachanensis is distinguished from C. albiramea by having elliptic leaves with leaf aspect ratio less than 2.5 (vs. oblong-lanceolate leaves with leaf aspect ratio more than 2.5).
To apply names to the species of Cryptocarya in Kaeng Krachan National Park, we examined the images of the lectotype and isolectotype of C. amygdalina and noticed that the description of the fruit morphology of C. amygdalina by de Kok (2015) does seem to not agree with the type material of C. amygdalina. While de Kok (2015) described the fruit stalk of C. amygdalina as "red, strongly swollen when mature" and used this state to distinguish C. amygdalina from morphologically similar species in the key, the type of C. amygdalina has fruit stalks not or only slightly swollen, as in our collection T3090. On the other hand, the fruit stalks of C. kaengkrachanensis are somewhat swollen, and brownish rather than red. The fruit stalks of C. scortechinii Gamble in Malay Peninsula are red and strongly swollen (e.g. G. Kedah, T. Witmore FRI 4683, KEP!). We suggest that the concept of C. amygdalina adopted by de Kok (2015) is a heterogeneous one that includes C. amygdalina s. str., C. scortechinii (see below) and C. kaengkrachanensis. In fact, the following specimen cited under C. amygdalina by de Kok (2015) is identical to C. kaengkrachanensis in leaf morphology; Phrae: between Ban Nam Krai and Pha Tuem, 16 Apr 1970, Smitinand T, Cheke A.S. 10817 [BKF 46511]).
Before concluding that T2069 was an undescribed species, we needed to compare it with the type material of C. floribunda Nees and C. scortechinii Gamble, two names that were treated as synonyms of C. amygdalina by de Kok (2015). The type specimens of C. floribunda [Wallich Cat. n. 2593, BM000880687, K000768399, MEL2390468, MEL2390469, MEL2390467, MNHN-P-P02010447] have only flowers and we cannot verify the fruit characters. However, these specimens are most similar to the lectotype and isolectotype of C. amygdalina in floral and vegetative morphology. Thus, we agree with the earlier treatment of de Kok (2015, 2016a) that C. floribunda is a synonym of C. amygdalina. We collected a specimen (T4796) morphologically similar to the type specimens of C. scortechinii [King's collector 6297, L0036248-lectotype, MEL2386583-isolectotype] at the elevation of 322 m at Khao Luang, peninsular Thailand. Although our collection is sterile, it is identified as C. scortechinii based on its leaf size, shape, and venation as well as its distribution in peninsular Thailand. As is shown in Fig. 5 and 6, C. scortechinii was placed in a distant position from C. amygdalina. Thus, our evidence does not support the treatment of de Kok (2015, 2016a) that C. scortechinii is a synonym of C. amygdalina. Cryptocarya kaengkrachanensis is easily distinguished from C. scortechinii by its elliptic leaves (aspect ratio lower than 2.5) that are obtuse at the apex and not lustrous above. Based on the evidence provided above, we here concluded that C. kaengkrachanensis is an undescribed species.
While de Kok (2015) included C. amygdalina in the group characterized by "Mature lower leaf surface glabrous, except on veins", both C. amygdalina and C. kaengkrachanensis have minute hairs on the lower surface of leaves that are almost invisible to the naked eye and hand lens (10 ×), but clearly visible under magnification (25 ×). Be-cause most species of Cryptocarya are more or less hairy on the lower blade surface and hairiness of leaves is very variable, we do not use the hairiness trait in the following key.
In his key, de Kok (2015) characterized C. amygdalina as "Tertiary veins scalariform" and other species as "Tertiary veins reticulate to scalariform", but the specimen T3090 of C. amygdalina has undulate scalariform veins that are connected by finer reticulate veins. Among Thai species of Cryptocarya, C. chanthaburiensis Kosterm., C. concinna Hance and C. densiflora Blume are characterized by reticulate tertiary veins, but other species including C. amygdalina and C. kaengkrachanensis have more or less scalariform tertiary veins connected with finer reticulate veins. Thus, we used only two categories of venations, reticulate and scalariform, in the key that follows. In de Kok (2015), C. diversifolia Blume, C. ferrea Blume, C. laotica (Gagnep.) Kosterm., C. nitens (Blume) Koord. & Valeton, and C. rugulosa Hook.f. from Thailand, but these species are not included in the following key because we could not confirm the distribution of these species in Thailand.