Taxonomic implications from morphological and anatomical studies in the section Stenodiptera from the genus Grammosciadium (Apiaceae)

Abstract Grammosciadium pterocarpum subsp. bilgilii and Grammosciadium pterocarpum subsp. sivasicum from Turkey are herein described as two new subspecies, and the species Grammosciadium schischkinii is synonymied under Grammosciadium pterocarpum subsp. pterocarpum. Quantitative variation of morphological and anatomical characters have been analysed to provide discriminative characters between the taxa of section Stenodiptera and to provide a key to the species. The taxonomic status of the taxa has been discussed in light of these morphological and fruit anatomical data using multivariate statistics such as MANOVA and Principal Component Analysis. The results are also used to present a critical discussion of characters used to distinguish and determine different taxa within Grammosciadium. MANOVA showed that ten characters, except stylopodium and style length, differed significantly among the taxa, and the results were confirmed by Tukey tests and PCA analysis (except the character of fruit number). However, only ranges of the characters of sepal length, fruit length, fruit width, fruit width/wing width ratio, and width of fruit wing are not overlapped. Qualitative characters of petiolate stipular segments of lower leaves and presence of funicular oil ducts in transvers section of mericarps were found as diagnostic characters.


Introduction
Th e genus Grammosciadium DC. is a taxonomically diffi cult group of taxa within tribe Scandiceae, subtribe Careae (Apiaceae) (Spalik et al. 2001, Spalik and Downie 2007, Ajani et al. 2008. It falls within the "apioid superclade" (Spalik and Downie 2007) and is closely related with Fuernrohria K.Koch, Carum L. and other taxonomically critical taxa. As an example, in particular the genus Carum has been recently shown to be polyphyletic in its current circumscription, and its various members are even found in diff erent tribes of subfamily Apioideae (Zakharova et al. 2012). Th e stem group age of Careae is of about 22 million years and has a center of origin in the Irano-Turanian region (Banasiak et al. 2013), and a long-term and spatio-temporarely shared evolutionary history of the various lineages is likely. Accordingly, taxonomy and systematics of these groups of taxa are still unsolved, because morphological characters often display high levels of homoplasy, and phylogenetic inference is scarce (e.g. Downie et al. 2010, Zakharova et al. 2012. Th e genus Grammosciadium is actually considered to be represented by two subgenera (Grammosciadium and Caropodium (Stapf & Wettst.) Tamamsch. & V.M.Vinogr.), which are further split into six sections in total (Bani and Koch 2015); however, this has been done with limited available pyhlogenetic evidence so far and applying an extreme taxanomically splitting concept above the species level. Subgenus Caropodium has been further splitted into two sections, namely sects. Caropodium and Stenodiptera (Koso-Pol.) Tamamsch. & V.M.Vinogr. (for a detailed overview refer to Bani and Koch 2015).
Th e section Stenodiptera of the genus Grammosciadium DC. has typical mericarps with winged lateral ribs. Additional important characters are the presence of erect stems, white fl owers, and 4-6-pinnatisect, narrowly linear-elliptic leaves. Th e section Stenodiptera morphologically resembles section Caropodium because of the winged fruits, which are absent in other members of the genus Grammosciadium (Tamamschian and Vinogradova 1969a, b, 1970, Vinogradova 1995. However, the section Caropodium mainly diff ers from section Stenodiptera by its more branched and distinctly sulcate stems (Hedge and Lamond 1972).
Both sections are also anatomically diff erent from each other: section Caropodium has funicular oil ducts in transvers section of mericarps (funicular oil ducts absent in section Stenodiptera) (Tamamschian and Vinogradova 1969a, b, 1970, Vinogradova 1995. Also fruit surface ornamentations is diff erent in both sections (Bani et al. 2016). Since both of these sections are included in subgenus Caropodium of genus Grammosciadium in the actual literature (Tamamschian and Vinogradova 1969a, b, 1970, Vinogradova 1995, we still follow the concepts of sections and subgenera for pragmatic reasons, but being fully aware that this is an artifi cially splitting concept and is awaiting phylogenetic analysis (work in progress).
Th e section Stenodiptera has three species which are distributed mainly in Turkey, and are additionally found in adjacent areas of Anatolia and Iran (Hedge and Lamond 1972, Vinogradova 1970, Bani and Koch 2015. All members are characteristic plants of the Irano-Turanian phytogeographic region (Takhtajan 1986, Hedge and Lamond 1972, Tamamschian 1987, Bani and Koch 2015. Among them, Grammosciadium schischkinii (V.M.Vinogr. & Tamamsch.) V.M.Vinogr. and G. haussknechtii Boiss. are endemic to Turkey and the third species, G. pterocarpum Boiss., can be regarded as subendemic plant with smaller distribution ranges outside Turkey (Vinogradova 1970, Pimenov and Leonov 2004, Pimenov and Sutory 2014. After the fi rst record of the genus Grammosciadium has been provided (Candolle 1829) Boissier (1844Boissier ( , 1872 Freyn (1901) published a subspecies of G. pterocarpum from Turkey, namely subsp. longipes. Th e respective type material provides only fl owering material and no fruits are available, and, therefore, this taxon is currently accepted as synonym of G. pterocarpum (Pimenov and Sutory 2014). Koso-Poliansky established Stenodiptera Koso-Pol. with all these three winged species as an independent genus (Koso-Poliansky 1914. Moreover he divided his genus Stenodiptera into two sections with Euryptera including S. pterocarpa (Boiss.) Koso-Pol. and Eustenodiptera including S. haussknechtii (Boiss.) Koso-Pol. and S. platycarpa (Boiss. & Hausskn.) Koso-Pol., which is mostly based on breadth of wings of mericarps (Koso-Poliansky 1914. Although a species named S. armena Bordz., which was collected from Turkey, was published by Bordzilowski (1915), Koso-Poliansky (1916) synonymised this species under S. haussknechtii one year later. According to Schischkin (1923) Koso-Poliansky's idea of establishing a new genus with the winged members was appropriate, but he added and highlighted an important taxonomic aspect: if a new genus has been established with winged fruited species separated from genus Gramosciadium sensu Boissier, then this must be with the earlier published name Caropodium rather than Stenodiptera. Hence, he reduced the genus Stenodiptera into synonymy of the genus Caropodium and also re-established C. armenum (Bordz.) Schischkin on species rank (Schischkin 1923). Vinogradova and Tamamschian (1968) accepted that "C. armenum is identical to C. pterocarpum (Boiss.) Schischkin" and they also described C. pterocarpum var. schischkinii V.M.Vinogr. & Tamamsch. as a new taxon based on a specimen collected from Turkey in 1916. Th is variety was distinguished by its broader and more undulated wings of the fruits (Vinogradova and Tamamschian 1968). Later Vinogradova (1969b and1970) reduced Caropodium to the rank of a subgenus and recognized section Stenodiptera including the taxa G. pterocarpum, G. pterocarpum var. schischkinii and G. haussknechtii within this subgenus. In Flora of Turkey, Hedge and Lamond (1972) presented G. pterocarpum var.
schischkinii and G. haussknechtii as synonyms of G. pterocarpum. Finally, Vinogradova (1995) increased G. schischkinii to species rank and also G. haussknectii was accepted as independent species again.
In summary, there are fi ve taxa in section Stenodiptera that have been described so far: Grammosciadium pterocarpum, G. haussknechtii, G. pterocarpum subsp. longipes Freyn, G. armenum and G. schisckinii. However, for more than a whole century botanists are wondering of how to distinguish and how to classify them.
Th e taxa are morphologically very similar to each other and original descriptions are often based on insuffi cient material (Grammosciadium haussknechtii, G. schisckinii and G. pterocarpum subsp. longipes only known from the types, G. armenum is known from the type and some very few additional individuals).
According to the most recent treatments, Grammosciadium pterocarpum, G. haussknechtii and G. schischkinii are currently accepted as distinct species and the other taxa of G. armenum and G. pterocarpum subsp. longipes were synonymised under G. pterocarpum (Vinogradova 1995, Pimenov and Sutory 2014, Bani and Koch 2015. Numerous specimens were collected from the whole distribution areas, which refl ect the morphological and presumably also genetic variation limits of the taxa of section Stenodiptera in Turkey for the purpose of a phylogenetic-taxonomic revision of the members of the whole genus Grammosciadium between the years of 2011 and 2014. We observed a large number of intermediate forms during our fi eld work. Moreover, we encountered many problems during the identifi cation process of the specimens. Th e previous diagnostic characters mostly overlapped and some of the populations and specimens were not identifi ed unambiguously. Additionally two populations were discovered recently from Turkey (one is from Sivas province, the other one is from Eskişehir province), and although they are very similar to Grammosciadium pterocarpum in terms of their habits, these populations do not match with available species descriptions.
Th e aims of this study were (1) to examine quantitatively pattern of morphological variation of the members of section Stenodiptera based on a representative and population-based sampling with 133 individuals from 17 populations in total, (2) to determine diagnostic morphological and anatomical characters for correct discrimination of the putative taxa, and (3) to provide a taxonomic concept for the newly discovered morphotypes.

Materials and methods
133 specimens (individuals) from the members of section Stenodiptera were examined and used as operational taxonomic units (OTUs) in the multivariate analyses. Gramosciadium pterocarpum (102 individuals from 14 populations), G. haussknechtii (11 individuals from one population; because it is known from only one locality), and populations of two new subspecies collected from Eskişehir (B.Bani 6983) and Sivas (B.Bani 6985) provinces of Turkey (10 individuals from one population, respectively). Th ese specimens, which are Table 1. Morphological characters and their statistics (mean ± standard deviation (SD), min-max range and range with 95% CI: confi dence interval) for the four groups (N: number of individuals used for morphological measurements). Characters that diff er signifi cantly (P<0.05) among the taxa as shown by MANOVA are marked with asterisks. Superscript letters indicate the results of Tukey tests, with taxa in the same homogeneous subset (P< 0.05) sharing the same letter.  listed in the Suppl. material 1 and marked with asteriks, include the types of two taxa (G. haussknechtii and G. schischkinii see Table 1) and refl ected the morphological variability exhibited by the species and populations from throughout its geographic range ( Figure  1). Characters used in the multivariate analyses were based on previous taxonomic treatments and our own examination of collected specimens. 12 quantitative characters were selected (Table 1). Characters were scored at the same developmental stage on each plant (fruiting stage). Measurements were taken from the best developed infructescence available on a given specimen. Th ree data sets were constructed and analyzed: (1) a matrix which included all specimens (133 OTUs and 12 characters), (2) and (3) were created by excluding the OTU's of G. haussknechtii and the new subpecies (collected from Eskişehir) from data set 1, respectively. Th e MANOVA was performed with IBM SPSS Statistics for Windows, Version 20.0. (Armonk, NY), using "Type III sum of squares", and was followed by "Tukey tests" using the harmonic mean sample size to determine patterns of signifi cant diff erences between the taxa. Th e F-test was used to determine which, if any, characters diff ered signifi cantly among the taxa studied. Th e PCA was conducted also using IBM SPSS Statistics for Windows, Version 20.0. (Armonk, NY). Identical parameters and procedures were used for all analyses on the three diff erent data sets.

Statistical analysis
Descriptive statistics for the 12 morphological characters are presented in Table 1. Th e MANOVA showed that 10 characters, except stylopodium and style length, diff ered signifi cantly (P < 0.05) among the taxa and newly discovered populations, and this was confi rmed by Tukey tests (Table 1). Furthermore, only ranges of the characters of sepal length, fruit length, fruit width, Fruit width/wing width ratio, width of fruit wing are not overlapped. Other six characters are overlapped (Table 1).
Etymology. We dedicate this new subspecies in memory of our dear colleague Dr. Bilgehan Bilgili who passed away in 2015. Diagnosis. Th e new subspecies is similar to Grammosciadium pterocarpum subsp. pterocarpum and G. platycarpum, but mainly diff ers from G. pterocarpum subsp. pterocar- pum by the fruits with two oil ducts in funiculus, and it diff ers from Grammosciadium platycarpum by its shorter fruits (0.9-1.1 cm, not 1.2-1.8 cm) and lower leaf sheaths without distinctly stalked stipular segments.
Etymology. Th e epithet of this new subspecies derived from Sivas province of Turkey where this taxon is distributed.
Primary leaf segments of basal leaves are shown in Figure 3. Grammosciadium haussknechtii is distinctly diff erent with the linear oblong leaf segments. G. platycarpum, G. pterocarpum subsp. bilgilii and one population of G. pterocarpum subsp. pterocarpum (B.Bani 6966) have lanceolate primary segments. Th e others do have ovatelanceolate to orbicular leaf shapes. Stipular segments which are shown in Figure 4 are quite similar each other except stipular segments of G. platycarpum which are long pedicellate. As shown in Figure 5, the fruits are ordered from narrowly winged to broadly winged one. Th e width of fruit wings is very highly variable character. It is impossible to distinguish G. schischkinii with wider fruit wings which was previously used as diagnostic character. Th is species has clearly similar fruits with fruits of subsp. ptero- carpum. G. haussknechtii, G. platycarpum and G. pterocarpum subsp. bilgilii and subsp. sivasicum have relatively narrow winged fruits than the fruits of subsp. pterocarpum. Figure 4 present the fruit anatomical structure of all taxa in the section Stenodiptera. G. platycarpum and G. pterocarpum subsp. sivasicum share similar character of presence of two funicular oil ducts in transverse section of mericarps ( Figure 6).

Discussion
Historically the species have been distinguished by a combination of quantitative and qualitative characters (Boissier 1844, 1872, Freyn 1901, Bordzilowski 1915, Vinogradova and Tamamschian 1968, Tamamschian and Vinogradova 1969, 1969a, 1970, Vinogradova 1995, Bani and Koch 2015, Bani et al. 2016. Boissier (1844) described Grammosciadium pterocarpum with the following diagnosis based on Aucher's specimen (with immature fruits) collected from Turkey: stem 8-13 cm, fi brous collar present at base, leaves 4.5×0.6 cm and resembling the leaves of Carum verticillatum W.D.J.Koch. Fruits are nearly 1 cm long, and fruit wings are 1.5 mm broad. G. haussknechtii: stems are 30 cm long, leaves are 8 cm long and less than 0.4 cm broad, fruits are 1-1,2 cm long. G. haussknechtii is close to G. pterocarpum, but it diff ers by its narrower leaves, narrower wings of fruits and shorter calyx teeth (Boissier 1872). Th is species known only from type material located with various herbaria (WU, E, K, LE). G. pterocarpum subsp. longipes was described by Frey in 1901 based on the specimens which were collected by Kronenburg from Van province in Turkey in 1889 (Freyn 1901). Th e lectotype was designated by Pimenov and Sutory (2014) from the herbaria of BRNM (lectotype) and WU (isolectotype). Th e diagnostic characters are as follows: large pointed calyx teeth and longer pedicels (these specimens have longer pedicels in contrast to the other specimens of G. pterocarpum) (Freyn 1901). Th is subspecies is currently under synonymy of G. pterocarpum (Pimenov and Sutory 2014). According to Bordzilowski (1915) in its original diagnosis G. armenum is close to G. pterocarpum and G. haussknechtii. It diff ers from both, by its broader leaves, shorter fruits and marginate stylopodium. It slightly diff ers from G. pterocarpum by larger stature, rotundate fruit apex (not truncate) and narrower wings of mericarps, and it diff ers from G. haussknechtii in having broader wings of fruits. Th e type of this species is deposited in KW and few vouchers are fund in LE herbarium. G. schischkinii is close to G. pterocarpum but diff ers from it in having more undulated and broader fruit wings (2.5-3.5 mm not 1.5-2 mm), fewer number of fruits (1-4 compared to 4-9), and a more branched stem (Vinogadova 1995). Th is species has been described based on one specimen only and is kept in LE herbarium. Th ere is no any other collection.
Although the characters of fruit number, width of fruit wings and undulation of wings have been used previously to distinguish Grammosciadium schischkinii from the other species (Vinogradova 1995), our data demonstrates that the measurements obtained from the type specimen of G. schischkinii clearly overlapped with characters of G. pterocarpum subsp. pterocarpum (Table 1). Also undulation of fruit wings is common in nearly all populations. We also achieved similar results for G. armenum, which has been previously recognized as synonym of G. pterocarpum. We did not fi nd any qualitative or quantitative character to distinguish these species. Th e type of G. pterocarpum subsp. longipes is also identical with subsp. pterocarpum. Length of calyx teeth and length of pedicels, which has been used as diagnostic characters (Freyn 1901) are overlapping with the other taxa and do not allow reliable diff erentiation. It is obvious from this study that suffi cient fruiting and fl owering material is needed for its proper taxonomic treatment.
MANOVA demonstrated that most of the characters diff er statistically among the groups (Table 1), and the range values of the various characters (sepal length, fruit length, fruit width, fruit width/wing ratio and width of fruit wings) can be used to distinguish the various taxa signifi cantly. Fruit length and sepal length separate Grammosciadium haussknechtii from subsp. sivasicum and subsp. bilgilii. Fruit width is distinguishing between G. haussknechtii and subsp. bilgilii. G. haussknechtii is clearly diff erent from the all others by its higher Fruit width/wing width ratio also separates subsp. bilgilii from subsp. pterocarpum. Another diagnostic character is width of fruit wings, which discriminates subsp. pterocarpum from all other taxa.
According to PCA on dataset 1 (complete dataset), individuals of G. haussknechtii are clearly distinguished from all other taxa as a distinct group mostly because of their larger ratio of fruit width/wing width (2-3,7 mm not 0,3-2 mm). Fruit length, fruit width/ length ratio, fruit width/wing ratio, fruiting pedicel and ray length are the most discriminative characters with the largest eigenvalues ( Figure 2A, Table 2). With PCA on dataset 2 (subsp. pterocarpum, subsp. sivasicum and subsp. bilgilii) individuals of subsp. bilgilii, are clearly distinguished from subsp. pterocarpum by the narrower wings of fruit (0.5-0.8 mm, not 1-3.1 mm). However, individuals of subsp. sivasicum are placed with an intermediate position between these two groups ( Figure 2B). Fruit length, fruit width/length ratio, width of fruit wing, fruiting pedicel, ray length, fruit number and ray number are the characters with highest eigenvalues (Table 2). Similarly, the PCA on the third dataset (subsp. pterocarpum and subsp. sivasicum) separates both taxa from each other, but few individuals of both groups are overlapping ( Figure 2C). PCA on dataset 2 and 3 show the same discriminative characters (except fruit number) ( Table 2). But as indicated above, subspecies sivasicum has two funicular oil ducts in the funiculus in transverse section of mericarps resembling a unique character within the section Stenodiptera.
Currently accepted taxa with the synonyms and a key to the members of section