Palmophyllum crassum, a New Record of an Ancient Species in Green Algae from Korea

Korean J. Environ. Biol. 35(3) : 319~328 (2017) https://doi.org/10.11626/KJEB.2017.35.3.319

INTRODUCTION

The green algae distributed in freshwater and seawater, even terrestrial habitats, are photosynthetic eukaryotes char- acterized by the presence of chloroplast with two envelope membranes, stacked thylakoids, and chlorophyll a and b with different morphological organizations such as mona- doid, palmelloid, coccoid, coenocytic, and filamentous (Pröschold and Leliaert 2007). This green lineage has been an essential member in the global ecosystem for a long time as the ancestor of land plants evolved by the early diver- gence of Streptophyta and Chlorophyta, from an ancestral green flagellate unicell (Leliaert et al. 2012). Therefore, it was generally recognized that the ancestral Chlorophyta is unicellular with the presence of flagella and organic body scales, but this concept has been a matter of debate (Leliaert et al. 2016). Recently, Zechman et al. (2010) challenged

that the earliest-diverging Chlorophyta comprises marine green algae with simple morphology by revealing a deep- branching clade which is a macroscopic algal group named as the order Palmophyllales including Palmophyllum, Ver- digellas and Palmoclathrus, based on the molecular phylo- genetic study.

The palmelloid green algae, Palmophyllum, Palmoclathrus and Verdigellas, thrive in deep seawater up to ^> 100 m deep, where has dimly light marine habitat (Womersley 1971;

Nelson and Ryan 1986; Ballantine and Aponte 1996). These seaweeds are characterized by a unique multicellularity, forming firm and well-defined macroscopic thalli, referred to as palmelloid thallus organization (Zechman et al. 2010).

Despite the lack of evidence of the systematic position within the green algae, these genera had been classified into the family Palmellaceae of the chlorophycean order Volvo- cales (Fritsch 1935), the family Palmellaceae of the order Tetrasporales (Womersley 1984), or the family Chlorophy- ceae of the order Chlorococcales (Nelson and Ryan 1984).

Recently, since the molecular phylogenetic assessment of

* Corresponding author: Myung Sook Kim, Tel. 064-754-3523, Fax. 064-756-3541, E-mail. [email protected]

ⓒ 2017. Korean Society of Environmental Biology.

Palmophyllum crassum, a New Record of an Ancient Species in Green Algae from Korea

Hyung Woo Lee and Myung Sook Kim*

Department of Biology, Jeju National University, Jeju 63243, Republic of Korea

Abstract - The continuous exploration in deep seawater from Korea makes us lead the discovery of ancient Chlorophyta, Palmophyllum, in the Korean coast. The phylogenetic analyses of 18S rRNA and rbcL genes demonstrate that our specimens are Palmophyllum crassum (Naccari) Rabenhorst, recorded in Japan and clearly distinguished from P. umbracola from New Zealand and California, USA. Palmophyllum crassum grows in the subtidal region, 8-30 m deep, and has a crustose thallus which is closely adherent to substrates such as non-geniculate crustose coralline algae, sponge, shells, or rocks. P. crassum is composed of numerous spherical cells embedded in the gelatinous matrix. The discovery of this ancient green seaweed implies that the Korean coast is one of the hotspots of algal species diversity and has the suitable marine environment for algal speciation. We suggest the grounds to conserve the Korean coast environmentally as the biodiversity center of marine species by studying the phylogeny of seaweeds.

Key words : 18S rRNA, molecular phylogeny, Palmophyllophyceae, Palmophyllum crassum, rbcL

<Original article>

the plastid-encoded atpB and rbcL genes and the nuclear- encoded 18S rRNA gene resolved that these palmelloid genera are positioned to the early diverged lineage in Chlo- rophyta forming monophyly in order level, the order Palmo- phyllales and the family Palmophyllaceae were established (Zeckman et al. 2010). Further, this early evolutionary lin- eage was elevated to the class Palmophyllophyceae, based on the chloroplast phylogenomic analyses (Leliaert et al.

2016).

The genus Palmophyllum is reported only two species, P.

crassum (Naccari) Rabenhorst and P. umbracola Nelson et Ryan, all over the world (Guiry and Guiry 2017). P. crassum has been known as the cosmopolitan species, unlike P.

umbracola, which is mainly restricted in the Southern hemisphere (Nelson and Ryan 1984). Although it has been recorded in Japan (Yoshida et al. 1990), several investiga- tions of Korean algal flora have not recognized P. crassum distributed in the deep region (Kang et al. 2011; Kang and Kim 2012).

Recently, the molecular methods have been applied to green seaweeds from Korean coasts to conduct taxonomic reexamination or to describe species diversity (Lee and Kim 2011, 2015). By continuous exploration of deep subtidal regions from Korean coastlines, we obtained crustose and firmly gelatinous green algal specimens unrecognized pre- viously and undertook the molecular and morphological

analyses. In addition, to compare with the entities from Korea, we carried out a collection in Japan. The aims of this study are to identify new green algal entities collected from deep subtidal and to resolve the species diversity of Korean green seaweed by reconstructing the green algal phylogeny with recognition of ancient lineage.

MATERIALS AND METHODS

The samples were collected using SCUBA (from depths of 8-30 m) in the southern part of Korea including Jeju Island and the central coast of Japan (Table 1). Underwater images were obtained using a digital camera (Stylus TG-4, Olympus, Japan). Samples were transported to the labora- tory and photographed by Canon Powershot G7X camera (Canon, Japan). After describing the external morphology, voucher specimens were made into pressed ones on the her- barium sheets. Samples for DNA analyses were detached from the thallus and dried via silica-gel. Samples for ana- tomical investigations were preserved in 5.0% formalin in seawater. Sections were done by a bench-top freezing micro- tome (NK-101-II; Nippon Optical Works Co., Ltd., Tokyo, Japan). Photomicrographs were obtained using a BX43 microscope (Olympus, Tokyo, Japan), with an EOS 600D digital camera (Canon, Japan). The digitized images were Table 1. Collection information and GenBank accession number of Palmophyllum crassum (Naccari) Rubenhorst specimens analyzed in this

study

Taxa Collection information Habit (depth/substratum) Voucher GenBank accession No.

18S rRNA rbcL

Palmophyllum crassum (Naccari) Rubenhorst

Gwideok 1-ri, Jeju, Korea;

31 May 2011 25-30 m deep / reef with

crustose coralline algae MSK-GA00830 - MF769365 Chujado, Jeju, Korea;

1 Oct. 2011 13-15 m deep / reef with

crustose coralline algae MSK-GA00915 MF769362 MF769367 Udo, Jeju, Korea;

1 Nov. 2011 18-25 m deep / reef with

crustose coralline algae MSK-GA00912 - MF769366 Udo, Jeju, Korea;

1 Nov. 2011 18-25 m deep / reef with

crustose coralline algae MSK131101-10 MF769361 MF769368 Udo, Jeju, Korea;

1 Nov. 2011 18-25 m deep / reef with

crustose coralline algae MSK131101-11 - MF769369 Hyeongjeseom, Busan, Korea;

28 Jul. 2016 10-15 m deep / reef with

sponge MSK160728-02 MF769363 -

Shimoda, Shizuoka, Japan;

17 Apr. 2017 8-15 m deep / reef with

crustose coralline algae MSK170417-01 MF769364 - Shimoda, Shizuoka, Japan;

17 Apr. 2017 8-15 m deep / reef with

sponge MSK170417-03 - MF769370

imported into Adobe Photoshop ver. 6.1 software (Adobe Systems Inc., San Jose, CA, USA). Voucher specimens were deposited in the herbarium of Jeju National University (JNUB), Korea and the National Institute of Biological Re- sources (NIBR), Incheon, Korea.

DNA was extracted using the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. Following DNA extraction, the polymerase chain reaction (PCR) were conducted to amplify the nuclear- encoded small subunit (SSU) 18S rRNA gene and the ribulose-1, 5-biphospate carboxylase/oxygenage (rbcL).

Amplification was performed in a 20-μL reaction, using AccuPower PCR PreMix (Bioneer, Daejeon, Korea) and Swift MaxPro thermal cyclers (ESCO, Singapore). The pri- mer and PCR protocols for SSU 18S rRNA were followed by Saunders and Moore (2013), and those for rbcL were followed by Heesch et al. (2009). PCR products were pu- rified using the AccuPrep Purification Kit (Bioneer) and sequenced commercially at Macrogen (Seoul, Korea).

Sequence edition and alignment were carried out using Chromas version 1.45 software (Queensland, Australia) and BioEdit software, respectively.

The uncorrected pair-wise distances were estimated using MEGA 5.0 software to assess the genetic variations in both SSU and rbcL sequences. The program Modeltest version 3.7 (Posada and Crandall 1998) was used to find the model of sequence evolution that best fit the data set by an Akaike Information Criterion (AIC). To construct both the SSU and rbcL phylogenetic tree, maximum likelihood (ML) analyses were performed using RAxML software (Stamatakis 2006).

RAxML was performed with all three codons partitioned and the GTR+Γ+I model. To identify the best tree, 200 independent tree inferences using the -# option with default - I (automatically optimized subtree pruning-regrafting re- arrangement) and -c (25 distinct rate categories) software options were performed. To generate bootstrap support values for the phylogeny, 1,000 bootstrap replicates were performed. The Bayesian phylogenetic inferences (BI) were generated using MrBayes ver. 3.1.2 software (Ronquist and Huelsenbeck 2003), and obtained the posterior probabilities using a Markov Chain Monte Carlo approach with checking by Tracer v1.6 (Rambaut et al. 2014). The ML and BI trees were edited with the program FigTree v1.4.0.

RESULTS

We analyzed a set of 41 SSU 18S rRNA gene sequences including the four Palmophyllum crassum (MF769361- MF769364) collected from Korea and Japan (Table 1), the representative taxa in charophytes, prasinophytes, and core chlorophytes with Palmophyllophyceae, Palmophyllum umbracola (FJ619275 and FJ619276) and Verdigella peltata (FJ619277 and LT174528). The SSU 18S rRNA data set consisted of 1897 characters. Among all sites, 758 (56.1%) were variable and 506 (26.7%) were parsimoniously infor- mative. The phylogenetic tree constructed by ML and BI was congruent, and we exhibited the ML topology including the bootstrap (BS) value in ML and posterior probability (PP) in BI (Fig. 1). Ulvophyceae-Trebouxiophyceae-Chlorophy- ceae (UTC) and Streptophyta formed each distinct clade in the SSU phylogenetic tree, but prasinophytes were polyphy- letic. Our four P. crassum specimens collected from Korea and Japan were identical, and they formed an independent clade with Palmophyllum umbracola from USA (FJ619275) and New Zealand (FJ619276), and Verdigellas peltata from Guadeloupe (FJ619277) and USA (LT174528) with strong supports of 100% in BS and 1.0 PP (Fig. 1). Palmophyllum crassum from Korean and Japanese collections showed 0.3% genetic divergences with P. umbracola and V. peltata, respectively. P. umbracola from New Zealand (FJ619276), the type locality, had 0.6% genetic divergences with P.

umbracola from California, USA (FJ619275). The Palmo- phyllophyceae clade was separated with other green algal taxa distinctly with strong supports (100% in BS, 1.00 in PP), exhibiting 13.7-26.3% pairwise distances with Strep- tophyta, 12.8-17.4% with prasinophytes, and 16.0-28.0%

with UTC clade. Especially, this Palmophyllophyceae clade was located at an earlier diverged position than other green seaweed lineages belonged to Ulvophyceae clade, such as Capsosiphon fulvelscens (EU099920), Urospora penicilli- formis (AB049417) and Ulva prolifera (EU099922), with 25.4-26.8% genetic divergences (Fig. 1).

To understand the phylogenetic relationships within the

class Palmophyllophyceae, we obtained six rbcL sequences

from Korean and Japanese specimens (MF769365-MF

769370) and aligned a set of 37 rbcL sequences from the

representative taxa in Ulvophyceae, Chlorophyceae, Tre-

bouxiophyceae and prasinophytes with two Streptophyta

species as outgroups ranging over 1272 sites in total. The variable position was 605 sites (47.6%) and the parsimony- informa tive was 543 sites (42.7%). We conducted both ML and BI analyses to construct the rbcL phylogenetic tree with BS and PP, and the topology was identical. We showed the rbcL phylogenetic tree derived from ML including BS and PP supports (Fig. 2). In the rbcL sequence analysis, the class Palmophyllophyceae was also monophyly and early- branched in Chlorophyta with powerful supports of 100%

BS and 1.0 PP. The genus Palmophyllum was clearly dis- tinguished from the genus Verdigellas with 4.0-5.9% inter- generic divergence. The Palmophyllum crassum specimens collected from Korea (MF769365-MF769369) were iden- tical with Japanese sequences from the Seto Inland Sea

(AP017927) supported in 99% BS and 1.0 PP, and showed 0.6% intraspecific divergence with a Japanese specimen (MF769370) from Shimoda, a Pacific coastal region of Japan. In the ML phylogenetic tree, P. crassum from Korea and Japan were distinguished from P. umbracola from New Zeanad and California, USA, with 1.4-5.3% interspecific divergence, exhibiting closer affinity with P. umbracola from New Zealand (EU586182) than one from California, USA (EU586180).

Class Palmophyllophyceae Lelaiaert et al., 2016 (국명신칭: 초록방석말강)

Order Palmophyllales Zechman et al., 2010 (국명신칭: 초록방석말목)

Fig. 1. The phylogenetic position of Palmophyllum crassum (Naccari) Rabenhorst from Korea and Japan in the maximum-likelihood (ML)

tree of Chlorophyta based on small subunit 18S rRNA sequences. The support values on each branch are from ML bootstrap (

50%,

left) and Bayesian posterior probability (

0.50, right). Scale bar represents: substitution/site.

Family Palmophyllaceae Zechman et al., 2010 (국명신칭: 초록방석말과).

Genus Palmophyllum Kützing, 1847 (국명신칭: 초록방석말속)

Palmophyllum crassum (Naccari) Rabenhorst 1868 (국명신칭: 초록방석말)

Basionym: Palmella crassa Naccari 1828.

Synonyms: Palmophyllum flabellatum Kützing 1845; Pal-

mophyllum gestroi Piccone 1879; Palmophyllum orbiculare Bornet ex Ardissone 1887; Palmophyllum crassum var. or- biculare (Bornet ex Ardissone) Feldmann 1937; Palmophyl- lum crassum var. typicum Feldmann 1937; Palmophyllum crassum f. gestroi (Piccone) Giaccone 1965.

Molecular vouchers: MF769361-MF769364 (SSU 18S rRNA); MF769365-MF769370 (rbcL).

Specimens examined: MSK-GA00830 (25-30 m deep,

Fig. 2. The phylogenetic relationships within the class Palmophyllophyceae in the maximum-likelihood (ML) tree of Chlorophyta based on

the plastid encoded rbcL gene sequences. Support values on each branch are from ML bootstrap (

50%, left) and Bayesian posterior

probability (

0.50, right). Scale bar represents: substitution/site.

Gwideok 1-ri, Jeju, Korea), MSK-GA00915 (13-15 m deep, Chujado, Jeju, Korea), MSK131101-09 & 11 (18-25 m deep, Udo, Jeju, Korea), MSK140722-18 & 19 (13-15 m deep, Seopseom, Jeju, Korea), MSK2014-v175 & 176 (15- 20 m deep, Udo, Jeju, Korea), MSK2014-v0216 (28-30 m deep, Dodu, Jeju, Korea), MSK2014-v0234 (14-15 m deep, Yongdam, Jeju, Korea), MSK160330-01 (21-24 m deep, Dodu, Jeju, Korea), MSK160527-s01 (21-24 m deep, Dodu, Jeju, Korea), MSK160627-01 & 02 (10-15 m deep, Geomundo, Yeosu, Korea), MSK160728-01, 02 & 03 (10- 15 m deep, Hyeonjeseom, Busan, Korea), MSK170417-01, 02 & 03 (8-15 m deep, Shimoda, Shizuoka, Japan), NIBR CL0000112650 (Gwideok 1-ri, Jeju, Korea).

Habit and anatomy: Plant grows in the subtidal zone, 8- 30 m deep. Thalli are crustose, closely adherent to substrate and not lobed, which forms and expands irregularly along the substrate plane (Fig. 3a-c). Thalli are usually distrib- uted on the shady vertical face of the reef. Thallus patches or sheets attach to non-geniculate crustose coralline algae, sponge, shells, or rocks. Thallus color is brilliant to dark green vividly (Fig. 3a-c). Thalli are unstructured and slight- ly firm gelatinous in texture, and the sizes range from 3- 7×4-8 cm to 10-13×15-18 cm, or largely expanded up to about 20×25 cm (Fig. 3a-f). Thalli thickness ranges from 643.87 μm to 893.92 μm. Thalli are composed of sphe rical cells, which are unstructured one another (Fig. 3g). In the transverse section, thalli are dorsiventral (Fig. 3g). From the dorsal surface to ventral region, the cell composition and density are changed. The cell density is greater at margins and dorsal surface than in central region and ventral side of thalli (Fig. 3g). The dorsal sided cells are overall greenish and 11.01-14.15 μm in diameter, except the outermost layer which is composed of little greenish cells, 6.13-7.81 μm in diameter (Fig. 3h). In the central and ven tral regions, cells are colorless, but dark green vacuoles and/or light green nucleus are conspicuous (Fig. 3i). These central and ventral cells are 9.92-12.74 μm in diameter.

DISCUSSION

The genus Palmophyllum Kützing was established on the basis of the report of P. flabellatum, having gelatinous-carti- laginous, flabellate, and foliose thallus composed of minute

cells by Kützing (1847). Rabenhorst (1868) transferred from Palmella crassa Naccari (1828) to Palmophyllum crassum, and described that P. crassum (Naccari) Rabenhorst is con- specific with P. flabellatum Kützing. Currently, P. crassum has been accepted as the generic type of Palmophyllum (Guiry and Guiry 2017). Feldman (1937), which treated P.

gestroi and P. orbiculare as synonyms of P. crassum, dis- cussed varieties of P. crassum dependent on the cell size and degree of attachment of the thallus to the substrate. Later, although Giaccone (1965) proposed species and varieties of Palmophyllum distributed in the Mediterranean Sea as forms of P. crassum depending on different environment, Cormaci et al. (2014) assessed that all varieties and forms of P. crassum were invalid on the grounds of the Interna- tional Code of Nomenclature (Melbourne Code, McNeill et al. 2012).

In the northwest Pacific region, P. crassum has solely been known in Japanese coastlines (Yoshida et al. 1990). The entities of P. crassum was firstly reported as P. orbiculare by Segawa (1936) from Miyake Island, Izu peninsula with the description of dark green thallus composed of spherical or oblong cells immersed in gelatinous substance, as well as adhering to the substratum such as rocks or calcareous al- gae on the comparison with figures of European specimens.

Later, Kajimura (1987) recorded another distribution of this species from Oki Islands, and Yoshida et al. (1990) listed it as P. crassum var. orbiculare.

Our molecular and morphological analyses of crustose green seaweed distributed in deep seawater resulted in the discovery of an ancient green seaweed, Palmophyllum cras- sum, from the Korean coast. The phylogenetic analysis of rbcL gene supported that the Korean specimens of P. cras- sum is identical to Japanese one (AP017927, Furukawa et al. 2017), which are distinguished from the type specimen of P. umbracola from New Zealand (EU586182, Zechman et al. 2010). The specimen of P. umbracola from California, USA (EU586180, Zechman et al. 2010) was not monophyly with the type specimen from New Zealand, exhibiting 5.6%

genetic divergence which is presumed that these two spec-

imens can be separated independently. Based on the rbcL

analysis, P. crassum known as a cosmopolitan species (Gui-

ry and Guiry 2017) should be reassessed from each local

specimen in the species level. Originally, P. crassum was

established based on specimens from the Mediterranean Sea

and the Adriatic Sea (Naccari 1828; Hauck 1885; Feldmann 1937; Cormaci et al. 2014). Therefore, world-wide exam- inations of P. crassum in rbcL analyses will lead to a better understanding of the ancient lineage of green seaweed in addition to recognition of species diversity.

Most green seaweeds distributed in deep water having dominant green light have the special composition of ca-

rotenoid pigments (Yokohama et al. 1977). These green seaweeds, referred to as deep-seawater type such as Clado- phora wrightiana, Codium minus and Umbraulva japonica, contain special chloroplast pigment: siphonixanthin or si- phonein which are efficient photosynthetic pigment under green illumination (Yokohama et al. 1977; Kageyama and Yokohama 1978). Palmophyllum species have not these Fig. 3. Palmophyllum crassum (Naccari) Rabenhorst. (a-c) Crustose habits in subtidal coral reef, which are tightly adherent to the substrate.

(a) The thallus growing in 25 m deep from Jeju Island, Korea, on 26th May 2016. (b) The thallus growing in 20 m deep from Busan, Korea, on 28th July 2016. (c) The thallus growing in 18 m deep from Shimoda, Japan, on 17th April 2017. (d-f) Voucher specimens of P. crassum. (d-e) MSK131101-10 & 11 collected subtidally in 18-25 m deep from Udo, Jeju, Korea on 1st November 2013. (f) MSK170417-03 collected subtidally in 8-15 m deep from Shimoda, Shizuoka, Japan. (g-i) Cross-section exhibiting coccoid cells embedded in a gelatinous matrix. (g) Transverse view of P. crassum, exhibiting dorsiventral structure. (h) Coccoid cells of dorsal side, entirely filled with greenish cytoplasm. (i) Coccoid cells of middle and ventral portion, showing light green colored nucleus and dark green colored vacuole (arrows), and cup-shaped chloroplast (arrowhead). Scale bars: (a)=3 cm; (b)=5 cm; (c)=6 cm; (d-f)=1 cm;

(g)=100 μm; (h)=20 μm; (i)=10 μm.

special chloroplast pigments (Yokohama et al. 1977), al- though they have been found in deep subtidal, growing on rock faces in the areas of shade, crevices and under rock overhangs (Nelson and Ryan 1986). In spite of a lack of siphonoxanthin or siphonein which functions as a photosyn- thetic pigment in deep seawater, it seems that Palmophyllum species have accommodated itself to dimly environment by maintaining increased chlorophyll b/a ratio (Sartoni et al.

1993). This increased chlorophyll b/a ratio may be regard- ed that early branching chlorophytes, like Palmophyllales lineage, have accumulated large quantities of chlorophyll b into core antennae of their photosystem to harvest blue-green light energy efficiently based on the molecular evolution of chlorophyllide a oxygenage in green plants (Kunugi et al.

2016). The ability of Palmophyllum to grow deeply may play an important role in the diminishment of abiotic stress and low competition for habitat (Zeckman et al. 2010).

In the rbcL analysis, Korean and Japanese specimens of P. crassum had closer affinity with P. umbracola from New Zealand than with Californian P. umbracola specimen supported weakly 57% in BS and 0.76 in PP (Fig. 2), de- spite quite high interspecific divergence, 4.6-5.3%. Recent studies also showed that some red seaweeds from subtidal, Amalthea and Psaromenia, have close intrageneric affinities between Korea and New Zealand (Lee et al. 2016). How- ever, Silva (1962) predicted the similarity of algal flora be- tween the northwestern and the northeastern Pacific based on the oceanic current, and he implied that there is bio- geographic barrier between both hemispheres. In the same manner, the study on the distribution of Ishige species from intertidal supported the biogeographic similarity on both sides of the northern Pacific (Lee et al. 2009). It is quite similar with the trans-Pacific zipper theory which describes a corresponding system of interlocking biogeographical sister areas (McCarthy 2003). Therefore, to reconsider the phylogenetic relationships of Palmophyllum species, it is necessary to examine more specimens known as P. crassum or P. umbracola from the Pacific region.

The northwestern Pacific region is recognized as a hot- spot of algal species diversity with the southern Australia region (Kerswell 2006). Our discovery of the green algal ancestor, Palmophyllum, from Korea seems to be good proof that the Korean coast is well qualified as one of the hotspots of algal species diversity. In addition, this implies that Kor-

ean coast provides a suitable environment for growing green seaweeds and further contributed the taxonomic radiation of green algae. Therefore, we should continuously try to ex- plore the green seaweed diversity in the Korean deep water and to conserve the oceanic environment of Korea as the biodiversity center of seaweed.

ACKNOWLEDGEMENT

We thank Dr. Wada at Shimoda Marine Research Center in University of Tsukuba for helpful cooperation to organize sampling in Shimoda, and also all members at the molec- ular phylogeny of marine algae laboratory in Jeju National University. This research was supported by a grant from the National Institute of Biological Resources (NIBR), funded by the Ministry of Environment (MOE) of the Republic of Korea (NIBR201701204).

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Received: 25 August 2017

Revised: 7 September 2017

Revision accepted: 8 September 2017