NEPETA MARGALLAICA (MENTHEAE: NEPETOIDEAE: LAMIACEAE): A NEW SPECIES FROM THE MARGALLA HILLS, PAKISTAN

A new species of Nepeta L. (Lamiaceae) from the Margalla Hills, in Pakistan, is described and illustrated. It belongs to Nepeta sect. Cataria sensu Bentham and is characterised by tuberous roots up to 3 cm in diameter, the presence of long multicellular flattened eglandular hairs, a spike-like inflorescence, an oblique calyx throat, unequal calyx teeth, and a 9 mm long white corolla. It is closely related to Nepeta cataria L., from which it differs in the root, indumentum, calyx and inflorescence characters. A distribution map of the new species is presented, as is an updated key to Nepeta sect. Cataria .


Introduction
Nepeta L. (Lamiaceae, Nepetoideae, Mentheae, Nepetinae) comprises approximately 300 species distributed in Southwest and Central Asia, Europe, North Africa and North America, with Southwest Asia and the Western Himalayas being the main centres of diversity (Acar et al., 2011).In the flora of Pakistan, Nepeta is the largest genus in the family Lamiaceae, comprising 55 species, 15 of which are endemic (Hedge, 1990;Jehan, 1996;Reshi et al., 2012;Sultan et al., 2021).In Pakistan, it is mainly recorded from Gilgit-Baltistan, Kashmir, Khyber Pakhtunkhwa, Balochistan, and northern and western Punjab.
Nepeta is a taxonomically complicated genus with morphological characters such as indumentum, calyx characters, leaf shape and size being highly variable even within a single species.Highlighting these taxonomic issues, Hedge (1990) noted that the genus contains both well-defined species and those that are exceedingly polymorphic.The lack of dependable morphological characters has contributed to the challenges in the delimitation of species in the genus.However, Hedge (1990) believed that nutlet size, shape, surface texture and areole length could provide more reliable characters.He also suggested that hybridisation and introgression probably play a role in the complexities of classification, and recommended comprehensive field studies and collation with the taxonomic situation 2 A new species of Nepeta from Pakistan in adjacent areas, particularly Kashmir, a rich area for Nepeta diversity in Pakistan (Hedge, 1990).
Previous infrageneric classifications of the genus based on morphological characters have been in conflict (Bentham, 1848;Briquet, 1896;Budantsev, 1993), largely due to differences in interpretation of the morphological variation of the characters used.However, molecular phylogenetic studies have greatly improved our knowledge of Lamiaceae and Nepeta (Jamzad et al., 2003;Li et al., 2016;Serpooshan et al., 2018;Zhao et al., 2021).
The results of molecular data analyses have supported the monophyly of Nepeta and the recognition of five major monophyletic groups within the genus, most of which included species from more than one section in previous classifications.These clades were: (i) sect.Spartonepeta, (ii) sects Macronepeta + Spicatae, (iii) sects Nepeta + Micranthae p.p. + Oxynepeta + Schizocalyx + Macrostegiae, (iv) sects Capituliferae + Denudatae + Micranthae p.p. + Micronepeta p.p., and (v) sect.Psilonepeta (Jamzad et al., 2003).These clades were supported by some floral characters, including corolla shape, bract texture, colour, and pollen exine ornamentation (Jamzad et al., 2003).The recent phylogenetic study in the subtribe Nepetinae (Serpooshan et al., 2018) showed that the genera Hymenocrater Fisch.& C.A.Mey., Lophantus Benth.and Marmoritis Benth.are all included in Nepeta, and under this assumption, the genus Nepeta is monophyletic.The various molecular phylogenetic studies support Bentham's classification in some groups, and highlight that monophyletic groups are not based on their annual or perennial habit, but on floral, inflorescence and other morphological characters.
The Margalla Hills, a mountain range situated on the northern edge of Islamabad, starts near Tret and ends near Taxila and is an extension of the Murree Hills (Western Himalayas).The range nestles between an elevation of 685 m at its western end and 1604 m at its eastern end; it is covered by deciduous and evergreen trees and supports a diverse shrub growth on the southern slopes and pines and oak groves with undergrowth of Myrsine africana L. on the northern slopes.Recent botanical expeditions to the Margalla Hills have provided new collections, among which were specimens with diagnostic characteristics that did not match those of known Nepeta species.
Here, we describe a new species of Nepeta collected from the Margalla Hills, and provide illustrations, scanning electron microscopy (SEM) images, images of root anatomical sections, and a distribution map for the new species.

Materials and methods
A field expedition to the Margalla Hills was undertaken, during which a population of a Nepeta species with tuberous roots was observed.Collected herbarium specimens of this species were studied in further detail at RAW (herbarium codes follow Thiers, continuously updated), using identification keys in the Flora of Pakistan (Hedge, 1990), Flora Iranica (Rechinger, 1982), Flora of China (Li & Hedge, 1994) and other relevant literature (Reshi et al., 2012), and were identified as belonging to a species new to science.
In addition to herbarium specimens, a tuberous root, detached from a healthy and mature plant, was examined to study its anatomical traits.The root was placed in formalin aceto-alcohol solution for 24 h.Thin serial transverse sections were then made using a Shandon microtome (Finesse 325; ThermoFisher Scientific, Waltham, MA, USA), using the paraffin wax method.Each section was passed through a series of 60%, 70% and 80% ethanol solutions and then stained following a method modified from that described by Mehdi et al. (2019), using 0.1% (w/v) toluidine blue, 0.5% (w/v) safranine O, and 0.2% (w/v) fast green solution.After staining, micrographs were taken of the root section at different magnifications, using a camera installed on the Primostar compound microscope (Zeiss 415500-0059-000; Oberkochen, Germany).Sections were labelled following the terminology of Moore (1987), and a full description of the root was produced following the terminology of Metcalfe & Chalk (1950).
Nutlets were removed from the herbarium specimens and studied directly under both a stereomicroscope (Olympus 605371; Tokyo, Japan) and a JEOL-5910 scanning electron microscope (Jeol, Tokyo, Japan) installed at the Centralized Resource Laboratory, Department of Physics, University of Peshawar.Material for examination by SEM was coated in gold and affixed to stubs, using double-sided adhesive tape.Based on the scanning electron micrographs, details of nutlet micromorphology were described, following Barthlott (1984) and Budantsev & Lobova (1997).
The conservation status of the species was assessed based on the IUCN Red List Criteria and Categories (IUCN, 2012).

Taxonomic treatment Species description
Nepeta margallaica A.    Distribution.So far known only from two localities approximately 1 km apart, in the Barkot area of the Margalla Hills (Figure 4).
Habitat and ecology.Found growing along maize field borders in the type locality.Flowering July to October, fruiting August to October.The aerial plant parts die back during the winter, and the plant resprouts in spring from the tuberous roots.
Etymology.The species is named after the Margalla Hills, where it was originally found.
Proposed IUCN conservation category.Both documented collections of this species originate from locations adjacent to a road, situated within a 2 km radius of each other, and near human settlements.The AOO and EOO are each 4 km 2 , it is recorded from two locations, there is evidence of a decline in at least the quality of habitat, and there are threats from overgrazing and uprooting by wild boars.As a precautionary measure, a provisional listing is provided as Critically Endangered (CR), based on B1ab(iii)+2ab(iii) criteria.Some plants are being maintained in the Botanical Conservatory at the National Agricultural Research Centre, Islamabad.Notes.Nepeta margallaica belongs to Nepeta sect.Cataria Benth.and to the N. cataria species group (Jamzad, 2012), whose members are characterised by having a cymose or paniculate inflorescence, consisting of distant verticillaster or close verticillasters, spike-like in the upper part; and oblique calyx throat, unequal teeth, middle lobe of the lower corolla lip dentate-crenate, with the margin turned upwards.Rather et al. (2012) have recorded Nepeta raphoanorhiza, known locally in Kashmir as kanz-gogal, as having an edible root; its edibility was also reported by Stewart (1972).The edibility of the tuberous roots of Nepeta margallaica should be investigated.

Nutlet micromorphology
In Nepeta, the micromorphological features of nutlets are important diagnostic characteristics.Previous studies have shown that there are two main types of exocarp ornamentation in Nepeta: smooth and sculptured (Jamzad et al., 2000).Nepeta margallaica, N. cataria and N. raphanorhiza nutlets belong to the smooth type.The SEM images of the nutlet surface showed detailed ornamentation of the smooth surface.The exocarp consists of rounded-irregular cells with flat outer periclinal walls, with some striation on cell surfaces.The anticlinal walls are raised, following the cell shape (Table , Figure 5).

Root anatomy
The root of Nepeta margallaica is more or less circular in outline in cross-section (Figure 6).The outer wall consists of indistinguishable rupture tissues forming the exodermis, behind which lies a 6-to 9-layered periderm composed of thick-walled polygonal cells.The cortex is generally composed of 18-32 layers of slightly irregular thick-walled parenchyma cells.The parenchyma in the cortical region has distinct starch grains with oval to oblong shapes, and it appears reddish.Cambium is generally indistinguishable, but to some extent, phloem fibers form a ring as a procambium (see Figure 6).The vascular ring is composed of phloem fibers and radially arranged xylem vessels, along with the medullary rays towards the centre.The tuberous root in Nepeta raphanorhiza has a spheroidal shape, in contrast to that of N. margallaica, which has a longer, carotiform tuber.We do not have anatomical data on the root of Nepeta raphanorhiza; here, the comparison between it and N. margallaica is based on their root morphological traits.
Like the new species, Nepeta connata (sect.Spicatae Benth.) also has a woody or tuberous rootstock.However, it has entire, linear to linear-lanceolate leaves; an 11-16 mm long calyx; and a mauve to blue-violet, 20-25 mm long corolla.The other species with tuberous roots is Nepeta tuberosa L. (sect.Pycnonepeta Benth.subsect.Subinterrupta Benth.) from the Iberian Peninsula, Morocco and Algeria, which has slender upright spikes of purple flowers in dense interrupted verticillasters with pinkish-grey bracts.All parts of the plant are covered with fine hairs, giving it a silvery appearance.

Figure 4 .
Figure 4. Map showing the distribution of Nepeta margallaica in Pakistan.

Figure 5 .
Figure 5. Scanning electron micrographs of Nepeta margallaica.A, Nutlet shape; B, nutlet surface; C and D, the exocarp, consisting of rounded-irregular cells with flat outer periclinal walls and raised anticlinal walls.All images of a nutlet from a pot-grown plant at the Botanical Conservatory of the National Agricultural Research Centre, taken by Abdullah Jan.

Table .
A comparison of diagnostic characters of Nepeta margallaica A.Sultan, Jamzad & A.Khan, sp.nov.with N. cataria and N. raphanorhiza